U.S. patent application number 15/316020 was filed with the patent office on 2021-11-04 for method and composition for determining specific antibody responses to species of filovirus.
The applicant listed for this patent is THE GOVERNMENT OF THE UNITED STATES, AS REPRESENTED BY THE SECRETARY OF THE ARMY, THE GOVERNMENT OF THE UNITED STATES, AS REPRESENTED BY THE SECRETARY OF THE ARMY. Invention is credited to Teddy KAMATA, Robert G. ULRICH.
Application Number | 20210341489 15/316020 |
Document ID | / |
Family ID | 1000005910268 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210341489 |
Kind Code |
A9 |
ULRICH; Robert G. ; et
al. |
November 4, 2021 |
METHOD AND COMPOSITION FOR DETERMINING SPECIFIC ANTIBODY RESPONSES
TO SPECIES OF FILOVIRUS
Abstract
The disclosure relates to compositions, assays, methods and kits
comprising one or more amino acid sequences of a filovirus protein,
or a fragment thereof, which find use in the detection of a
filovirus infection and/or the presence of antibodies specific for
a filovirus in a biological sample.
Inventors: |
ULRICH; Robert G.;
(Frederick, MD) ; KAMATA; Teddy; (Frederick,
MD) |
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Applicant: |
Name |
City |
State |
Country |
Type |
THE GOVERNMENT OF THE UNITED STATES, AS REPRESENTED BY THE
SECRETARY OF THE ARMY |
Fort Detrick |
MD |
US |
|
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20170089920 A1 |
March 30, 2017 |
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Family ID: |
1000005910268 |
Appl. No.: |
15/316020 |
Filed: |
June 3, 2015 |
PCT Filed: |
June 3, 2015 |
PCT NO: |
PCT/US15/34080 PCKC 00 |
371 Date: |
February 22, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62007195 |
Jun 3, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 2333/08 20130101; G01N 2469/20 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The invention was made with support from the National
Institute of Allergy and Infectious Diseases (R01AI96215), and the
Defense Threat Reduction Agency (contract CB3948).
Claims
1. A detection agent comprising one or more amino acid sequences of
a filovirus protein, or a fragment thereof, and a substrate,
wherein the one or more amino acid sequences of the filovirus
protein is attached to the substrate.
2. The detection agent of claim 1, wherein the one or more amino
acid sequences of a filovirus protein is from a filovirus selected
from Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus,
Reston ebolavirus, Bundibugyo ebolavirus, and Tai Forest
ebolavirus.
3. The detection agent of claim 1, wherein the one or more amino
acid sequences of a filovirus protein, or fragment thereof, is
selected from nucleoprotein (NP), virion protein 40 (VP40),
glycoprotein (GP), virion protein (VP35), virion protein (VP30),
virion protein (VP24), RNA-dependent RNA polymerase (L), or any
combination thereof.
4. The detection agent of claim 1, wherein the one or more amino
acid sequences of a filovirus protein comprises GP, or fragment
thereof.
5. The detection agent of claim 1, wherein the one or more amino
acid sequences of a filovirus protein, or fragment thereof,
comprises a mucin-like domain fragment of GP (GP-mucin).
6. The detection agent of claim 1, wherein the one or more amino
acid sequences of a filovirus protein, or fragment thereof,
comprises a GP ectodomain (GP.DELTA.TM).
7. The detection agent of claim 1, comprising at least three
different amino acid sequences of at least three different
filovirus proteins, or fragments thereof.
8. The detection agent of claim 7, wherein the at least three
different proteins comprise NP, VP40, and GP, or fragments
thereof.
9. The detection agent of claim 8, wherein NP comprises a protein
having at least 90% sequence identity to the sequence selected from
the group consisting of SEQ ID NO:4 (Zaire NP); SEQ ID NO:10 (Sudan
NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Tai Forest NP);
SEQ ID: 28 (Reston NP); and SEQ ID NO: 34(Marburg NP); VP40
comprises a protein having at least 90% sequence identity to the
sequence selected from the group consisting of SEQ ID NO: 2 (Zaire
VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40);
SEQ ID 20 (Tai Forest VP40); SEQ ID NO: 26 (Reston VP40); and SEQ
ID NO: 32 (Marburg P40); and GP comprises a GP-mucin domain having
at least 90% sequence identity to the sequence selected from the
group consisting SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12
(Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO:
24 (Tai Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ
ID NO: 36 (Marburg GP-mucin).
10. The detection agent of claim 8, wherein NP is selected from the
group consisting of SEQ ID NO:4 (Zaire NP); SEQ ID NO:10 (Sudan
NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Tai Forest NP);
SEQ ID: 28 (Reston NP); and SEQ ID NO: 34(Marburg NP); VP40 is
selected from the list consisting of SEQ ID NO: 2 (Zaire VP40); SEQ
ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID 20
(Tai Forest VP40); SEQ ID NO: 26 (Reston VP40); SEQ ID NO: 32
(Marburg VP40); and GP comprises a GP-mucin domain selected from
the group consisting of SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO:
12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID
NO: 24 (Tai Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and
SEQ ID NO: 36 (Marburg GP-mucin).
11. The detection agent of any of claims 1-10, wherein the
substrate is selected from the group consisting of a microarray,
microparticles, and nanoparticles.
12. The detection agent of any of claims 1-10, wherein the
substrate is a microarray.
13. The detection agent of any of claims 1-10, wherein the one or
more amino acid sequences of a filovirus protein is provided as a
recombinant protein or a fragment thereof.
14. A method for detecting the presence of filovirus-specific
antibody in biological sample obtained from a subject comprising:
(a) incubating the biological sample with a detection agent
comprising one or more amino acid sequences of a filovirus protein,
or a fragment thereof, attached to a substrate under conditions
that allow binding of the filovirus-specific antibody to the
detection agent; and (b) detecting the filovirus-specific antibody
bound to detection agent.
15. The method of claim 14, wherein the filovirus is selected from
the group consisting of Marburg marburgvirus, Sudan ebolavirus,
Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Tai
Forest ebolavirus.
16. The method of claim 14, wherein the one or more amino acid
sequences of a filovirus protein, or a fragment thereof, comprises
nucleoprotein (NP), virion protein 40 (VP40), glycoprotein (GP),
virion protein (VP35), virion protein (VP30), virion protein
(VP24), RNA-dependent RNA polymerase (L), or any combination
thereof.
17. The method of claim 16, wherein the one or more amino acid
sequences of a filovirus protein, or a fragment thereof, comprises
GP.
18. The method of claim 16, wherein the one or more amino acid
sequences of a filovirus protein, or a fragment thereof, comprises
a mucin-like domain fragment of GP (GP-mucin).
19. The method of claim 16, wherein the one or more amino acid
sequences of a filovirus protein, or a fragment thereof, comprises
a GP ectodomain (GP.DELTA.TM).
20. The method of claim 14, wherein the detection agent comprises
at least three different amino acid sequences of at least three
different filovirus proteins, or fragments thereof.
21. The method of claim 20, wherein the three different proteins,
or fragments thereof, comprise NP, VP40, and GP.
22. The method of claim 21, wherein NP comprises a protein having
at least 90% sequence identity to the sequence selected from the
group consisting of SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO: 16; SEQ
ID NO: 22; SEQ ID: 28; and SEQ ID NO: 34; VP40 comprises a protein
having at least 90% sequence identity to the sequence selected from
the group consisting of SEQ ID NO: 2; SEQ ID NO: 8; SEQ ID NO: 14;
SEQ ID 20; SEQ ID NO: 26; and SEQ ID NO: 32; and GP comprises a
GP-mucin domain having at least 90% sequence identity to the
sequence selected from the group consisting of SEQ ID NO: 6; SEQ ID
NO: 12; SEQ ID NO: 18; SEQ ID NO: 24; SEQ ID NO: 30; and SEQ ID NO:
36.
23. The method of claim 21, wherein NP is selected from the group
consisting of SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO: 16; SEQ ID NO:
22; SEQ ID: 28; and SEQ ID NO: 34; VP40 is selected from the group
consisting of SEQ ID NO: 2; SEQ ID NO: 8; SEQ ID NO: 14; SEQ ID 20;
SEQ ID NO: 26; and SEQ ID NO: 32; and GP comprises a GP-mucin
domain selected from the group consisting of SEQ ID NO: 6; SEQ ID
NO: 12; SEQ ID NO: 18; SEQ ID NO: 24; SEQ ID NO: 30; and SEQ ID NO:
36.
24. A method for identifying a subject infected with a filovirus,
comprising: determining whether a filovirus-specific antibody is
present in a sample obtained from the subject, wherein the
determining comprises: (a) incubating the biological sample with a
detection agent comprising one or more amino acid sequences of a
filovirus protein, or a fragment thereof, attached to a substrate
under conditions that allow binding of the filovirus-specific
antibody to the detection agent; and (b) detecting the
filovirus-specific antibody bound to the detection agent, wherein
the detection of the filovirus-specific antibody identifies that
the subject is infected with a filovirus.
25. The method of claim 24, wherein the filovirus is selected from
the group consisting of Marburg marburgvirus, Sudan ebolavirus,
Zaire ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Tai
Forest ebolavirus.
26. The method of claim 24, wherein the at least one protein or
fragment thereof comprises nucleoprotein (NP), virion protein 40
(VP40), glycoprotein (GP), virion protein (VP35), virion protein
(VP30), virion protein (VP24), RNA-dependent RNA polymerase (L), or
any combination thereof.
27. The method of claim 26, wherein the at least one protein or
fragment thereof comprises GP.
28. The method of claim 26, wherein the at least one protein or
fragment thereof comprises a mucin-like domain fragment of GP
(GP-mucin).
29. The method of claim 26, wherein the at least one protein or
fragment thereof comprises a GP ectodomain (GP.DELTA.TM).
30. The method of claim 24, wherein the method comprises contacting
the sample with a combination of three different proteins, or
fragments thereof, from the filovirus.
31. The method of claim 30, wherein the three different proteins,
or fragments thereof comprise NP, VP40, and GP.
32. The method of claim 31, wherein NP comprises a protein having
at least 90% sequence identity to the sequence selected from the
group consisting of SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO: 16; SEQ
ID NO: 22; SEQ ID: 28; and SEQ ID NO: 34; VP40 comprises a protein
having at least 90% sequence identity to the sequence selected from
the group consisting of SEQ ID NO: 2; SEQ ID NO: 8; SEQ ID NO: 14;
SEQ ID 20; SEQ ID NO: 26; and SEQ ID NO: 32; and GP comprises a
GP-mucin domain having at least 90% sequence identity to the
sequence selected from the group consisting of SEQ ID NO: 6; SEQ ID
NO: 12; SEQ ID NO: 18; SEQ ID NO: 24; SEQ ID NO: 30; and SEQ ID NO:
36.
33. The method of claim 31, wherein NP is selected from the group
consisting of SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO: 16; SEQ ID NO:
22; SEQ ID: 28; and SEQ ID NO: 34; VP40 is selected from the group
consisting of SEQ ID NO: 2; SEQ ID NO: 8; SEQ ID NO: 14; SEQ ID 20;
SEQ ID NO: 26; and SEQ ID NO: 32; and GP comprises a GP-mucin
domain selected from the group consisting of SEQ ID NO: 6; SEQ ID
NO: 12; SEQ ID NO: 18; SEQ ID NO: 24; SEQ ID NO: 30; and SEQ ID NO:
36.
34. A method for identifying whether a subject is infected with a
filovirus, comprising: determining whether an antibody to the
filovirus is present in a sample obtained from the subject, wherein
the determining comprises: (a) contacting the sample with at least
one protein, or a fragment thereof, from the filovirus to which the
antibody can specifically bind; and (b) detecting specific binding
between the at least one protein and the antibody, wherein the
detection of specific binding identifies that the subject is
infected with a filovirus.
35. The method of claim 34, wherein the filovirus is selected from
the group consisting of Marburg marburgvirus, Sudan ebolavirus,
Zaire ebolavirus, Reston ebolavirus, bugyo ebolavirus, and Tai
Forest ebolavirus.
36. The method of claim 34, wherein the at least one protein or
fragment thereof comprises nucleoprotein (NP), virion protein 40
(VP40), glycoprotein (GP), virion protein (VP35), virion protein
(VP30), virion protein (VP24), RNA-dependent RNA polymerase (L), or
any combination thereof.
37. The method of claim 36, wherein the at least one protein or
fragment thereof comprises GP.
38. The method of claim 36, wherein the at least one protein or
fragment thereof comprises a mucin-like domain fragment of GP
(GP-mucin).
39. The method of claim 36, wherein the at least one protein or
fragment thereof comprises a GP ectodomain (GP.DELTA.TM).
40. The method of claim 34, wherein the method comprises contacting
the sample with a combination of three different proteins, or
fragments thereof, from the filovirus.
41. The method of claim 40, wherein the three different proteins,
or fragments thereof comprise NP, VP40, and GP.
42. The method of claim 41, wherein NP comprises a protein having
at least 90% sequence identity to the sequence selected from the
group consisting of SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO: 16; SEQ
ID NO: 22; SEQ ID: 28; and SEQ ID NO: 34; VP40 comprises a protein
having at least 90% sequence identity to the sequence selected from
the group consisting of SEQ ID NO: 2; SEQ ID NO: 8; SEQ ID NO: 14;
SEQ ID 20; SEQ ID NO: 26; and SEQ ID NO: 32; and GP comprises a
GP-mucin domain having at least 90% sequence identity to the
sequence selected from the group consisting of SEQ ID NO: 6; SEQ ID
NO: 12; SEQ ID NO: 18; SEQ ID NO: 24; SEQ ID NO: 30; and SEQ ID NO:
36.
43. The method of claim 41, wherein NP is selected from the group
consisting of SEQ ID NO:4; SEQ ID NO:10; SEQ ID NO: 16; SEQ ID NO:
22; SEQ ID: 28; and SEQ ID NO: 34; VP40 is selected from the group
consisting of SEQ ID NO: 2; SEQ ID NO: 8; SEQ ID NO: 14; SEQ ID 20;
SEQ ID NO: 26; and SEQ ID NO: 32; and GP comprises a GP-mucin
domain selected from the group consisting of SEQ ID NO: 6; SEQ ID
NO: 12; SEQ ID NO: 18; SEQ ID NO: 24; SEQ ID NO: 30; and SEQ ID NO:
36.
44. The method of claim 34, comprising the detection agent of claim
1.
45. A kit comprising the detection agent of claim 1; at least one
reagent that can detect a filovirus-specific antibody bound to the
detection agent of claim 1; and instructions for use of the
kit.
46. A method for making the detection agent of claim 1 comprising:
expressing one or more recombinant polynucleotide sequences
encoding an amino acid sequence of a filovirus protein, or a
fragment thereof in an expression system; and fixing the encoded
amino sequence of a filovirus protein, or a fragment thereof, on a
surface of the substrate.
47. The method of claim 46, wherein the expression system comprises
a prokaryotic cell, a eukaryotic cell, or in vitro translation, or
any combination thereof.
48. The method of claim 47, wherein the prokaryotic cell comprises
E. coli.
49. The method of claim 47, wherein the eukaryotic cell is selected
from the group consisting of yeast, an insect cell, and a mammalian
cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
application No. 62/007,195 filed on Jun. 3, 2014, which is
incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0003] This application is submitted with a Sequence Listing text
file that serves as both the computer readable form (CRF) and the
paper copy required under 37 C.F.R. .sctn.1.821. The sequence
listing, which is incorporated by reference, includes a the
entitled "SequenceListing.txt" which is 92 kilobytes in size and
was created on Jun. 1, 2015.
BACKGROUND
[0004] Filoviruses, which include marburgviruses and ebolaviruses,
cause severe viral hemorrhagic fever. The first outbreak of Marburg
virus was recorded in 1967 in Germany and Yugoslavia, and was
traced to infected African green monkeys from Uganda (1). Since
then, major outbreaks of marburgviruses have occurred in
sub-Saharan Africa. The first outbreaks of Ebola virus were
documented in Sudan and The Democratic Republic of Congo in 1976
(2, 3). Because no licensed therapeutics or vaccines are currently
available, cycles of filovirus outbreaks are a major concern in
biodefense as well as public health. Filoviral hemorrhagic fever is
characterized by rapid disease onset and mortality rates of up to
90% (4). Following an incubation period that can range from 2-21
days, infected patients commonly develop non-specific flulike
symptoms of fever, vomiting, loss of appetite, headache, abdominal
pain, fatigue, and diarrhea, while bleeding occurs in a smaller
number of infections (1, 3, 5). Case fatalities are associated with
reduced adaptive immune responses (6, 7) and the release of high
levels of immune response mediators (8-10) that contribute to
vascular dysfunction, coagulation disorders, shock and eventual
multi-organ failure (2).
[0005] There is a persistent need for sensitive and reliable
serological approaches for examining filoviral infections. Because
genetic material from the pathogen is often missing, antibody
detection methods are indispensable, especially for examining
nonviremic patients and for disease surveillance. While ELISAs for
detecting specific IgG and IgM based on live virus preparation were
previously developed (11-13), the need for BSL-4 labs and
associated safety issues are major limitations. Serological assays
based on recombinant filovirus antigens are alternatives that do
not require infectious agents, and several ELISAs were reported
(14-18). For example, Nakayama and coworkers developed a GP-based
ELISA representative of all six species of filoviruses and analyzed
human patient sera from Ebola and Marburg virus outbreaks (Nakayama
et al, 2010). However, these previous methods have only addressed a
limited number of antigens and species of filoviruses. The
Filoviridae family includes one species of Marburg virus (Marburg
marburgvirus), with five species of Ebola virus (Sudan, Zaire,
Reston, Bundibugyo, and Tai Forest ebolavirus) that are each a
cause of severe hemorrhagic fevers in primates including humans
(2). Further complicating assay development, the single-stranded,
negative-sensed RNA genome (.about.19 kB) encodes seven structural
proteins (1, 19, 20) that are each potential antigens: the
nucleoprotein (NP), virion protein 35 (VP35), VP40, glycoprotein
(GP), VP30, VP24, and RNA-dependent RNA polymerase (L). Major
functions of each component of the viral proteome were previously
characterized. The RNA genome is encapsulated by NP, and the
ribonucleoprotein complex is associated with VP35, VP30, and L (21,
22). Transcription and replication of the viral genome requires L,
NP, and VP35 (23), while transcription for Ebola virus, but not
Marburg virus, requires VP30 as an additional co-factor (24, 25).
VP40 is a matrix protein critical for virion assembly as well as
budding from infected cells (26, 27), and VP24 appears to play a
role in nucleocapsid assembly and inhibition of interferon
signaling (28-30). Unlike Marburg GP, Ebola GP is expressed
following RNA editing, while the unedited transcript encodes a
soluble GP that is released from infected cells (31, 32). Further,
trimeric GP complexes on the virion surface are receptors for
fusion and entry into the host cell (33-35).
[0006] Compositions and methods that can provide for a fast,
accurate, and comprehensive serological analysis would help
faciliate identification and diagnosis of filovirus infection
(e.g., one or more filovirus antibodies in a sample) in the general
human population as well as potentially animal populations. Such
compositions and methods would provide an important tool in the
detection, management, and containment of filovirus outbreaks, and
ultimately help to reduce mortality rates and public panic that are
associated with these hemorrhagic fever viruses.
SUMMARY
[0007] In one aspect the disclosure provides a detection agent
comprising one or more amino acid sequences of a filovirus protein,
or a fragment thereof, and a substrate, wherein the one or more
amino acid sequences of the filovirus protein is attached to the
substrate.
[0008] In embodiments, the one or more amino acid sequences of a
filovirus protein is from a filovirus selected from Marburg
marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston
ebolavirus, Bundibugyo ebolavirus, and Tai Forest ebolavirus. In
some embodiments the one or more amino acid sequences of a
filovirus protein, or fragment thereof, is selected from
nucleoprotein (NP), virion protein 40 (VP40), glycoprotein (GP),
virion protein (VP35), virion protein (VP30), virion protein
(VP24), RNA-dependent RNA polymerase (L), or any combination
thereof of the same or different filovirus. In some embodiments the
detection agent comprises from two or more amino acid sequences to
twenty or more amino acid sequences (e.g., 2, 3, 4, 5, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more amino
acid sequences) from the same or different filovirus. In some
embodiments the detection agent comprises at least three different
amino acid sequences of at least three different filovirus
proteins, or fragments thereof, and in further embodiments, the at
least three different proteins may comprise NP, VP40, and GP, or
fragments thereof. In some embodiments the detection agent
comprises a protein having at least 90% sequence identity to the
sequence of a filovirus protein.
[0009] In some embodiments, the detection agent may comprise a
substrate is selected from the group consisting of a microarray,
microparticles, and nanoparticles, and such substrates may be made
of materials including glasses, plastics, chemical/biological
polymers, metal (magnetic and non-magnetic) semiconductors,
ceramics, and the like. In some embodiments the detection agent is
a microarray.
[0010] In certain embodiments, the one or more amino acid sequences
of a filovirus protein may be provided as a recombinant protein or
a fragment thereof.
[0011] In an aspect the disclosure provides a method for detecting
the presence of filovirus-specific antibody in biological sample
obtained from a subject comprising: [0012] (a) incubating the
biological sample with a detection agent comprising one or more
amino acid sequences of a filovirus protein, or a fragment thereof,
attached to a substrate under conditions that allow binding of the
filovirus-specific antibody to the detection agent; and [0013] (b)
detecting the filovirus-specific antibody bound to detection
agent.
[0014] In another aspect the disclosure provides a method for
identifying a subject infected with a filovirus, comprising:
[0015] determining whether a filovirus-specific antibody is present
in a sample obtained from the subject, wherein the determining
comprises: [0016] (a) incubating the biological sample with a
detection agent comprising one or more amino acid sequences of a
filovirus protein, or a fragment thereof, attached to a substrate
under conditions that allow binding of the filovirus-specific
antibody to the detection agent; and [0017] (b) detecting the
filovirus-specific antibody bound to the detection agent,
[0018] wherein the detection of the filovirus-specific antibody
identifies that the subject is infected with a filovirus.
[0019] In a further aspect, the disclosure provides a method for
identifying whether a subject is infected with a filovirus,
comprising:
[0020] determining whether an antibody to the filovirus is present
in a sample obtained from the subject, wherein the determining
comprises: [0021] (a) contacting the sample with at least one
protein, or a fragment thereof, from the filovirus to which the
antibody can specifically bind; and [0022] (b) detecting specific
binding between the at least one protein and the antibody,
[0023] wherein the detection of specific binding identifies that
the subject is infected with a filovirus.
[0024] In various embodiments of the above aspects relating to
methods, the filovirus may be selected from the group consisting of
Marburg marburgvirus, Sudan ebolavirus, Zaire ebolavirus, Reston
ebolavirus, Bundibugyo ebolavirus, and Tai Forest ebolavirus or any
combination thereof. In embodiments the methods comprise one or
more amino acid sequences of a filovirus protein, or a fragment
thereof, comprises nucleoprotein (NP), virion protein 40 (VP40),
glycoprotein (GP), virion protein (VP35), virion protein (VP30),
virion protein (VP24), RNA-dependent RNA polymerase (L), or any
combination thereof.
[0025] In some embodiments the detection agent comprises from two
or more amino acid sequences to twenty or more amino acid sequences
(e.g., 2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or more amino acid sequences) from the same or
different filovirus or filovirus protein. In some embodiments the
detection agent comprises at least three different amino acid
sequences of at least three different filovirus proteins, or
fragments thereof, and in further embodiments, the at least three
different proteins may comprise NP, VP40, and GP, or fragments
thereof. In some embodiments the detection agent comprises a
protein having at least 90% sequence identity to the sequence of a
filovirus protein.
[0026] In further embodiments, the methods can comprise the
comparison the amount of an filovirus-specific antibody detected
according to the method with one or more control values (e.g.,
measuring the amount of signal generated from incubating the
detection agent with a normal (healthy and/or uninfected)
biological sample).
[0027] In other aspects the disclosure provides a kit comprising,
the detection agent as described herein; at least one reagent that
can detect a filovirus-specific antibody bound to the detection
agent; and instructions for use of the kit.
[0028] In yet a further aspect, the disclosure provides a method
for making the detection agent described herein, the method
comprising: [0029] expressing one or more recombinant
polynucleotide sequences encoding an amino acid sequence of a
filovirus protein, or a fragment thereof in an expression system;
and [0030] fixing the encoded amino sequence of a filovirus
protein, or a fragment thereof, on a surface of the substrate.
[0031] In embodiments of this aspect, the method may comprise an
recombinant expression system including a prokaryotic cell, a
eukaryotic cell, or in vitro translation, or any combination
thereof. In further embodiments, the prokaryotic cell may comprise
a bacterium such as, for example, E. coli. In other embodiments,
the eukaryotic cell may be selected from the group consisting of
yeast, an insect cell, and a mammalian cell.
[0032] Other aspects and embodiments will be apparent to those of
skill in the art in view of the description and illustrative
Examples that follow.
BRIEF DESCRIPTION OF THE DRAWING
[0033] FIG. 1A-1C. Validation of filovirus microarray using control
antibodies. A panel of antibodies against A) Marburg virus, B)
Zaire ebolavirus, and C) Sudan ebolavirus proteins were tested on
printed microarrays. All antibodies are mouse monoclonal except for
anti-Zaire-NP and -VP40 which are both rabbit polyclonal. Bound
antibodies were detected fluorescently on a microarray scanner.
Background-corrected fluorescence intensities were averaged across
technical replicates on the microarrays. Bars represent mean
fluorescence (RFU).+-.SEM. All GP .DELTA.TMs were expressed in
insect cells except for Marburg-GP .DELTA.TM (Musoke) which was
expressed in mammalian cells. Bundibugyo (B), Tai Forest (T),
Marburg (M), Reston (R), Sudan (S), and Zaire (Z).
[0034] FIG. 2A-2C. IgG antibody response detected using filovirus
microarray. Naive, post-immunization (immunized), and post-viral
challenge (challenged) sera from A) Zaire ebolavirus and B) Marburg
virus animal studies were applied to assembled microarrays. Bound
IgG antibodies were detected fluorescently on a microarray scanner.
Following data pre-processing, normalized fluorescence signals were
averaged across the five animals in each study. Bars represent
normalized mean fluorescence (RFU).+-.SEM. The cutoff line
represents two standard deviations above the mean antibody signal
observed in naive sera. For each antigen-antibody response, paired
t-test was done for naive versus immunized and naive versus
challenged sera. Unless indicated with an `*`, all immunized and
challenged samples above the cutoff line were found to have
significant antibody increases (p<0.05) in comparison with the
naive samples. Bundibugyo (B), Tai Forest (T), Marburg (M), Reston
(R), Sudan (S), and Zaire (Z). C) Side-by-side comparison of
GP-specific IgG signals in challenged sera from Zaire ebolavirus
and Marburg marburgvirus studies.
[0035] FIG. 3A-B. Comparison of antibody signals between E. coli-
and eukaryotic cell-expressed GP. Data was acquired and analyzed in
a similar manner as in FIG. 2. Bars represent normalized mean
fluorescence (RFU).+-.SEM. All GP-mucins were expressed in E. coli.
All GP .DELTA.TM were expressed in insect cells except for Marburg
GP .DELTA.TM (Musoke) which was expressed in mammalian cells. A)
Zaire ebolavirus study. B) Marburg marburgvirus study.
[0036] FIG. 4A-B. Microarray schematic and representative scanned
fluorescent image of processed microarray. A) Slide schematic shows
multiple 12.times.12 microarrays printed on a slide, along with an
enlarged layout of an individual microarray. Table below provides
the sample identity for each triplicate microarray spots. Red
circles represent fluorescently-labeled streptavidin serving as
reference markers for orientation purposes. Bundibugyo (B), Tai
Forest (T), Marburg (M), Reston (R), Sudan (S), and Zaire (Z). B)
Representative GenePix.RTM.-scanned image of microarray processed
with naive (left) and post-challenge sera (right) from the ZEBOV
vaccine study.
[0037] FIG. 5. Scatter plot of individual antibody responses for
primates challenged with Zaire ebolavirus. Bound IgG were detected
fluorescently on a microarray scanner. Background-corrected
fluorescence intensities were averaged across technical replicates
on the microarrays. Pre-vaccination, dark circles;
post-vaccination, open circles; post-viral challenge, dark
triangles. Bundibugyo (B), Tai Forest (T), Marburg (M), Reston (R),
Sudan (S), and Zaire (Z).
[0038] FIG. 6. Scatter plot of individual antibody responses for
primates challenged with Marburg marburgvirus. Bound IgG were
detected fluorescently on a microarray scanner.
Background-corrected fluorescence intensities were averaged across
technical replicates on the microarrays. Pre-vaccination, dark
circles; post-vaccination, open circles; post-viral challenge, dark
triangles. Bundibugyo (B), Tai Forest (T), Marburg (M), Reston (R),
Sudan (S), and Zaire (Z).
[0039] FIG. 7. IgM signals for sera collected from a single animal
in the ZEBOV challenge groups. Bound antibodies were detected
fluorescently on a microarray scanner. Background-corrected
fluorescence intensities were averaged across technical replicates
on the microarrays. Bundibugyo (B), Tai Forest (T), Marburg (M),
Reston (R), Sudan (S), and Zaire (Z).
[0040] FIG. 8A-C. Phylogenic relationships between filovirus
strains based on amino acid sequences of NP and GP mucin-like
domain. Separate dendrograms representative of sequence similarity
between filovirus strains were derived based on amino acid
sequences of A) a conserved region of 406 residues of NP (BDBV,
TAFV, RESTV, SUDV, EBOV-residues 20-425; MARV-residues 2-407), and
B) the highly unconserved mucin-like domain of GP, consisting of 33
residues at the N-terminus of the domain region (BDBV, TAFV,
RESTV-residues 2-34; EBOV and MARV-residues 1-33). Maximum
likelihood tress are shown with bootstrap values (out of 1000
replicates) shown at internal nodes. (C) Sequence identity matrix
comparison of filovirus NP and GP mucin-like domain amino acid
sequences. Full length sequences of NP and the GP mucin-like domain
were used to generate percent identity matrices. NP (light grey,
upper triangle) is highly conserved among ebolavirus strains and
more divergent from MARV, while the GP mucin-like domain is highly
variable and exhibits minimal sequence identify among all filovirus
strains examined.
[0041] FIG. 9. Antibody reactivity to filoviral proteins in a
cohort of ebola and marburg survivors. Heat map displaying IgG
reactivity associated with filoviral infection and controls.
Hierarchical clustering by Euclidean distance average linkage
method was used to visualize protein microarray results. Normalized
and log2-transformed data was applied for creating the heat map.
The IDs of proteins are listed in the rows (*insect and**mammalian
expressed), the cells represent individual sera samples, and the
survivor and control groups are listed on the bottom of the colored
bars. The blue bars show ACAM2000 healthy controls, the purple bars
healthy controls from Uganda and the green bars three MARV, SUDV
and BDBV survivor groups.
[0042] FIG. 10. Ebola and Marburg survivors convalescent IgG
responses to autologous GP-mucin, NP and VP40 recombinant proteins.
Panel a shows reactivity to BDBV, panel b to SUDV-Gulu and panel c
to MARV antigens by survivors. The filled circles denote survivors
and open circles controls. Each circle corresponds to individual
sera sample and the red line represents geometric mean of all
samples in each group. Statistical analysis was performed using
Prospector software for comparing survivor Vs. controls.
Significant differences between the two groups in terms of p values
are shown as *p,0.05, **p,0.01, ***p,0.001
[0043] FIG. 11. Survivor sera antibody cross-reactivity to
filoviral heterologous compared to autologous antigens. Each bar
represents the mean antibody binding of all samples in a survivor
group (MARV, SUDV or BDBV) expressed as relative fluorescence units
(RFU).
[0044] FIG. 12. Antibody responses by a replication defective
recombinant EBOV/SUDV GP vaccinated subject to filoviral proteins.
The bars represent mean values of four replicates (* insect and **
mammalian expressed).
[0045] FIG. 13. Comparison antibody responses to GP proteins.
[0046] FIG. 14. List of significant antibody responses to
autologous and heterologous antigens and their p-values. The three
survivor groups are on the left column and the antigen hits are
listed on the right cells.
DETAILED DESCRIPTION
[0047] As discussed in further detail below, the inventors have
developed compositions of matter (e.g., detection agents, kits,
etc.) and methods that can provide for fast, accurate,
comprehensive and convenient (e.g., point-of-care assays) detection
of filovirus antibodies in biological samples such as, for example,
sera. The efficacy of the compositions and methods relating to the
general technology is demonstrated through the illustrative
embodiments disclosed in the Examples. For example, certain
embodiments provide for the preparation and use of protein
microarrays as the detection agent disclosed and described herein,
and by including one or more amino acid sequences of at least one
filovirus protein (e.g., GP or fragments thereof), or combinations
of filovirus proteins and/or fragments thereof (e.g., NP, GP, and
VP40, as discussed below), allow for the detection of an antibody
to one or more filovirus (e.g., Ebola and/or Marburg virus)
species. Further, and unexpectedly, the inventors have identified
that in particular embodiments one or more amino acid sequences of
filovirus proteins and/or fragments thereof, may be recombinantly
expressed in expression systems, including prokaryotic cells,
(e.g., E. coli) without any loss of filovirus-specific antibody
binding activity or specificity.
[0048] In practicing the technology disclosed herein, many
conventional techniques in molecular biology, protein biochemistry,
cell biology, immunology, and microbiology are used. These
techniques are well-known and are explained in, e.g., Current
Protocols in Molecular Biology, Vols. I-III, Ausubel, Ed. (1997);
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989); DNA Cloning: A Practical Approach, Vols. I and H, Glover,
Ed. (1985); Transcription and Translation, Hames & Higgins,
Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized
Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to
Molecular Cloning; the series, Meth. Enzymol., (Academic Press,
Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller
& Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and
Meth. Enzymol., Vols. 154 and 155, Wu & Grossman, and Wu, Eds.,
respectively. Methods to detect and measure the levels of protein
complexes are well-known in the art and include ELISA assays, and
co-immunoprecipitation assays.
[0049] All references referred to within the body of the
application are incorporated herein by reference in their
entirety.
[0050] Definitions
[0051] Virus as used herein refers to a small infectious agent that
replicates only inside the living cells of other organisms. Viruses
can infect all types of life forms, from animals and plants to
microorganisms, including bacteria and archaea. Virus particles
(known as virions) typically consist of two or three parts: i) the
genetic material made from either DNA or RNA, long molecules that
carry genetic information; ii) a protein coat that protects these
genes; and in some cases iii) an envelope of lipids that surrounds
the protein coat when they are outside a cell. The shapes of
viruses range from simple helical and icosahedral forms to more
complex structures. The average virus is about one one-hundredth
the size of the average bacterium.
[0052] Filoviruses generally refer to viruses of the viral family
called Filoviridae and infection can cause severe hemorrhagic fever
in humans and nonhuman primates. So far, three members of this
virus family have been identified: Marburgvirus, Ebolavirus, and
Cuevavirus. Five species of Ebolavirus have been identified: Tai
Forest (formerly Ivory Coast), Sudan, Zaire, Reston and Bundibugyo.
Ebola-Reston is the only known Filovirus that does not cause severe
disease in humans; however, it can still be fatal in monkeys and it
has been recently recovered from infected swine in South-east Asia.
Structurally, filovirus virions (complete viral particles) may
appear in several shapes, a biological feature called pleomorphism.
These shapes include long, sometimes branched filaments, as well as
shorter filaments shaped like a "6", a "U", or a circle. Viral
filaments may measure up to 14,000 nanometers in length, have a
uniform diameter of 80 nanometers, and are enveloped in a lipid
(fatty) membrane. Each virion contains one molecule of
single-stranded, negative-sense RNA. New viral particles are
created by budding from the surface of their hosts' cells; however,
filovirus replication strategies are not completely understood.
[0053] The genus Ebolavirus (or "Ebola" or "Ebola virus") is a
virological taxon included in the family Filoviridae, order
Mononegavirales. The members of this genus are generally referred
to as ebolaviruses. The five known virus species are named for the
region where each was originally identified: Bundibugyo ebolavirus,
Reston ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus
(originally Cote d'Ivoire ebolavirus), and Zaire ebolavirus.
[0054] The genus Marburgvirus (or "Marburg" or "Marburg virus")
refers to the species, Marburg marburgvirus, which includes two
known marburgviruses, Marburg virus (MARV) and Ravn virus (RAVV).
Both viruses cause Marburg virus disease, a form of hemorrhagic
fever, in humans and nonhuman primates.
[0055] Cuevavirus is a genus in the family Filoviridae that has one
identified species, Lloviu cuevavirus (LLOV or "cueva virus").
Studies indicate that LLOV is a distant relative of the more widely
known Ebola and Marburg viruses.
[0056] DNA is used herein (Deoxyribonucleic acid) is a molecule
that encodes the genetic instructions used in the development and
functioning of all known living organisms and many viruses. DNA is
a nucleic acid; alongside proteins and carbohydrates, nucleic acids
compose the three major macromolecules essential for all known
forms of life. Most DNA molecules consist of two biopolymer strands
coiled around each other to form a double helix. The two DNA
strands are known as polynucleotides since they are composed of
simpler units called nucleotides. Each nucleotide is composed of a
nitrogen-containing nucleobase--either guanine (G), adenine (A),
thymine (T), or cytosine (C)--as well as a monosaccharide sugar
called deoxyribose and a phosphate group. The nucleotides are
joined to one another in a chain by covalent bonds between the
sugar of one nucleotide and the phosphate of the next, resulting in
an alternating sugar-phosphate backbone. According to base pairing
rules (A with T and C with G), hydrogen bonds bind the nitrogenous
bases of the two separate polynucleotide strands to make
double-stranded DNA.
[0057] Proteins are large biological molecules, or macromolecules,
consisting of one or more long chains of amino acid residues (e.g.,
"peptide", "polypeptide", or "amino acid sequence"). Proteins
perform a vast array of functions within living organisms,
including catalyzing metabolic reactions, replicating DNA,
responding to stimuli, and transporting molecules from one location
to another. Structural differences in proteins typically arise
based on differences between secondary structure (primarily
sequence of amino acids), which is dictated by the nucleotide
sequence of their genes, and tertiary and/or quaternary structure
(protein folding, three-dimensional structure, and domain
interactions), all of which can determine activity.
[0058] An antigen (Ag), abbreviation of antibody generator, is any
structural substance which serves as a target for the receptors of
an adaptive immune response, TCR or BCR or its secreted form
antibody, respectively. Each antibody is specifically selected
after binding to a certain antigen because of random somatic
diversification in the antibody complementarity determining regions
(a common analogy used to describe this is the fit between a lock
and a key). In summary, an antigen is a molecule that binds to
Ag-specific receptors but cannot induce an immune response in the
body by itself. Antigen was originally a structural molecule that
binds specifically to the antibody, but the term now also refers to
any molecule or a linear fragment that can be recognized by highly
variable antigen receptors (B-cell receptor or T-cell receptor) of
the adaptive immune system.
[0059] An antibody (AB), also known as an immunoglobulin (Ig), is a
large, Y-shape protein produced by plasma cells that is used by the
immune system to identify and neutralize pathogens such as bacteria
and viruses. The antibody recognizes a unique molecule of the
harmful agent, called an antigen, via the variable region. Each tip
of the "Y" of an antibody contains a paratope that is specific for
one particular epitope (similarly analogous to a key) on an
antigen, allowing these two structures to bind together with
precision. Using this binding mechanism, an antibody can tag a
microbe or an infected cell for attack by other parts of the immune
system, or can neutralize its target directly (for example, by
blocking a part of a microbe that is essential for its invasion and
survival). The ability of an antibody to communicate with the other
components of the immune system is mediated via its Fc region
(located at the base of the "Y"), which contains a conserved
glycosylation site involved in these interactions. The production
of antibodies is the main function of the humoral immune
system.
[0060] A microarray as used herein refers to the technology
generally identified as a multiplex lab-on-a-chip. Typically a
microarray comprises a 2D array on a solid substrate (usually a
glass slide or silicon thin-film cell) that assays large amounts of
biological material using high-throughput screening miniaturized,
multiplexed and parallel processing and detection methods. A
protein microarray (or protein chip) is a high-throughput method
used to track the interactions and activities of proteins, and to
determine their function, and determining function on a large
scale. Its main advantage lies in the fact that large numbers of
proteins can be tracked in parallel. The chip consists of a support
surface such as a glass slide, nitrocellulose membrane, bead, or
microtitre plate, to which an array of capture proteins is bound.
Probe molecules, typically labeled with a fluorescent dye, may
added to the array. Any reaction between the probe and the
immobilized protein emits a fluorescent signal that is read by a
laser scanner. Protein microarrays may be rapid, automated,
economical, and highly sensitive, consuming small quantities of
samples and reagents. Methodology relating to protein microarrays
was introduced as early as 1983, illustrated using antibody-based
microarrays (also referred to as antibody matrix).
[0061] Mucin are a family of high molecular weight, heavily
glycosylated proteins (glycoconjugates) produced by epithelial
tissues in most organisms of Kingdom Animalia. Mucins' key
characteristic is their ability to form gels; therefore they are a
key component in most gel-like secretions, serving functions from
lubrication to cell signalling to forming chemical barriers. They
often take an inhibitory role. Some mucins are associated with
controlling mineralization, including nacre formation in mollusks,
calcification in echinoderms and bone formation in vertebrates.
They bind to pathogens as part of the immune system. Overexpression
of the mucin proteins, especially MUC1, is associated with many
types of cancer. Although some mucins are membrane-bound due to the
presence of a hydrophobic membrane-spanning domain that favors
retention in the plasma membrane, most mucins are secreted onto
mucosal surfaces or secreted to become a component of saliva.
[0062] The cell is the basic structural, functional, and biological
unit of all known living organisms. Cells are the smallest unit of
life that can replicate independently, and are often called the
"building blocks of life". A prokaryote is a single-celled organism
that lacks a membrane-bound nucleus (karyon), mitochondria, or any
other membrane-bound organelles. A eukaryote is any organism whose
cells contain a nucleus and other organelles enclosed within
membranes.
[0063] Recombinant protein is a protein produced by a recombinant
DNA that encodes for the protein sequence. Recombinant DNA (rDNA)
molecules are DNA molecules formed by laboratory methods of genetic
recombination (such as molecular cloning) to bring together genetic
material from multiple sources, creating sequences that would not
otherwise be found in biological organisms. Once a recombinant DNA
is inserted into bacteria, these bacteria will make protein based
on this recombinant DNA. This protein is known as "Recombinant
protein".
[0064] Infection is the invasion of an organism's body tissues by
disease-causing agents, their multiplication, and the reaction of
host tissues to these organisms and the toxins they produce.
Infectious disease, also known as transmissible disease or
communicable disease is illness resulting from an infection.
Infections discussed herein are typically caused filoviruses.
Detection Agent
[0065] In a general aspect, the disclosure provides a detection
agent comprising one or more amino acid sequences of a filovirus
protein, or a fragment thereof, and a substrate wherein the one or
more amino acid sequences of the filovirus protein is attached to
substrate. In certain embodiments, the one more amino acid
sequences of a filovirus protein may comprise a sequence of a
protein from a filovirus selected from Marburg marburgvirus, Sudan
ebolavirus, Zaire ebolavirus, Reston ebolavirus, Bundibugyo
ebolavirus, and Tai Forest ebolavirus.
[0066] In some embodiments, the one or more amino acid sequences of
a filovirus protein, or fragment thereof, may comprise a sequence
from a nucleoprotein (NP), virion protein 40 (VP40), glycoprotein
(GP), virion protein (VP35), virion protein (VP30), virion protein
(VP24), RNA-dependent RNA polymerase (L), or a fragment thereof, or
any combination thereof. Any amino acid sequence that provides for
binding and recognition of a filovirus specific antibody may be
used in connection with the detection agent. In some embodiments,
the amino acid sequence may exhibit little to no cross-reactivity
to filovirus specific antibodies that are directed to a particular
type of filovirus or a particular filovirus protein. In certain
embodiments the one or more amino acid sequences of a filovirus
protein comprises GP, or fragment thereof. The GP or fragment
thereof may comprise a mucin-like domain fragment of GP (GP-mucin)
or a GP ectodomain (GP.DELTA.TM). While the detection agent
comprises at least one amino acid sequence of a filovirus protein,
it may also comprise a plurality of such amino acid sequences. In
some embodiments the detection agent comprises from two or more
amino acid sequences to twenty or more amino acid sequences (e.g.,
2, 3, 4, 5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, or more amino acid sequences) that may be selected from the
same or different filovirus and/or the same or different filovirus
protein. As a further example, in some embodiments, such as those
illustrated in the non-limiting Examples, the detection agent may
include at least three different amino acid sequences of at least
three different filovirus proteins, or fragments thereof (e.g., NP,
VP40, and GP, or fragments thereof).
[0067] In some embodiments, the amino acid sequences comprising the
detection agent can comprise a sequence that is not identical to
the protein sequence from which it is derived. Some minor changes
in the primary amino acid sequence and/or post-translational
modification and processing of the sequence may be allowable as
long as the sequence modification does not interfere with the
ability of the filovirus-specific antibody to bind. In some
embodiments, the detection agent can comprise one or more amino
acid sequences having at least 90% sequence identity (e.g., 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the filovirus
protein from which it is derived. In some embodiments, the amino
acid sequences may comprise an NP sequence having at least 90%
sequence identity to the sequence selected from the group
consisting of SEQ ID NO:4 (Zaire NP); SEQ ID NO: 10 (Sudan NP); SEQ
ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Tai Forest NP); SEQ ID
NO: 28 (Reston NP); and SEQ ID NO: 34 (Marburg NP); a VP40 sequence
having at least 90% sequence identity to the sequence selected from
the group consisting of SEQ ID NO: 2 (Zaire VP40); SEQ ID NO: 8
(Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID NO: 20 (Tai
Forest VP40); SEQ ID NO: 26 (Reston VP40); and SEQ ID NO: 32
(Marburg VP40); and/or a GP-mucin domain having at least 90%
sequence identity to the sequence selected from the group
consisting SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12 (Sudan
GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO: 24 (Tai
Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ ID NO:
36 (Marburg GP-mucin). In further embodiments, the detection agent
may comprise an NP sequence selected from the group consisting of
SEQ ID NO: 4 (Zaire NP); SEQ ID NO: 10 (Sudan NP); SEQ ID NO: 16
(Bundibugyo NP); SEQ ID NO: 22 (Tao Forest NP); SEQ ID NO: 28
(Reston NP); and SEQ ID NO: 34(Marburg NP); a VP40 selected from
the list consisting of SEQ ID NO: 2 (Zaire VP40); SEQ ID NO: 8
(Sudan VP40); SEQ ID NO: 14 (Bundibugyo VP40); SEQ ID 20 (Tai
Forest VP40); SEQ ID NO: 26 (Reston VP40); SEQ ID NO: 32 (Marburg
VP40); and/or a GP-mucin domain selected from the group consisting
of SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID NO: 12 (Sudan GP-mucin);
SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ ID NO: 24 (Tai Forest
GP-mucin); SEQ ID NO: 30 (Reston GP-mucin); and SEQ ID NO: 36
(Marburg GP-mucin).
[0068] As discussed herein, in certain embodiments the substrate
may comprise a bead or particle (e.g., microparticle or
nanoparticle). In other embodiments the substrate may comprise a
substantially planar surface with a plurality of addressable
locations that are each associated with a known amino acid sequence
of a filovirus protein, and optionally one or more control
locations (e.g., a microarray). As discussed further, in some
embodiments, the one or more amino acid sequences of a filovirus
protein is provided as a recombinant protein or a fragment
thereof.
[0069] In certain embodiments, the detection agent can comprise any
one or more controls such as, for example, a positive control, a
negative control, an assay standard, an assay calibrator, a
competition assay ligand, a labeled peptide or a solid-phase
capture agent. Similarly in some embodiments the amino acid
sequences (including any controls as well as the amino acid
sequence(s) of a filovirus protein(s)) may comprise a synthetic
peptide, a recombinant polypeptide, a substantially purified
natural polypeptide, a peptide mimetic, an oligonucleotide aptamer,
a polypeptide aptamer, any fragment thereof that can be bound by a
filovirus-specific antibody and any combinations thereof. In
certain embodiments the detection agent comprises an amino acid
sequence of a filovirus protein that is recombinantly produced.
[0070] In certain embodiments the detection agent comprises a
protein microchip or microarray comprising one or more amino acid
sequences of a filovirus protein, or a fragment thereof, and a
substrate to which the one or more amino acid sequences are
attached. In these embodiments, the microarrays may be useful in a
variety of applications including large-scale and/or
high-throughput screening for a filovirus-specific antibody that
bind to the microarray. In other embodiments, the microarray can be
used to identify compound or agents that are capable of modulating
the interactions between the filovirus-specific antibody and the
amino acid sequence to which it binds.
[0071] Regardless of the particular format (e.g., microarray-based
or particle-based), the detection agent may be prepared according
to any of the techniques described herein or otherwise known in the
art. For example, in embodiments relating to a protein microarray,
the array can be prepared in a number of methods known in the art.
For example, glass microscope slides are treated with an
aldehyde-containing silane reagent. Small volumes of protein
samples in a phosphate-buffered saline with 40% glycerol may be
spotted onto the treated slides using a high-precision
contact-printing robot. After incubation, the slides are immersed
in a buffer containing for example, bovine serum albumin (BSA) to
quench the unreacted aldehydes and to form a BSA layer that
functions to prevent non-specific protein binding in subsequent
applications of the array/microchip. Alternatively, proteins or
protein complexes can be attached to a BSA-NHS slide by covalent
linkages. BSA-NHS slides are fabricated by first attaching a
molecular layer of BSA to the surface of glass slides and then
activating the BSA with N,N'-disuccinimidyl carbonate. As a result,
the amino groups of the lysine, aspartate, and glutamate residues
on the BSA are activated and can form covalent urea or amide
linkages with protein samples spotted on the slides. Alternatively,
arrays may be prepared as discussed in the illustrative Examples
below.
Methods
[0072] In further aspects, the disclosure relates to a number of
methods. In an aspect the disclosure provides a method for
detecting the presence of filovirus-specific antibody in biological
sample obtained from a subject including: (a) incubating the
biological sample with a detection agent comprising one or more
amino acid sequences of a filovirus protein, or a fragment thereof,
attached to a substrate under conditions that allow binding of the
filovirus-specific antibody to the detection agent; and (b)
detecting the filovirus-specific antibody bound to detection
agent.
[0073] In another aspect, the disclosure provides a method for
identifying a subject infected with a filovirus, comprising
determining whether a filovirus-specific antibody is present in a
sample obtained from the subject, wherein the determining includes:
(a) incubating the biological sample with a detection agent
comprising one or more amino acid sequences of a filovirus protein,
or a fragment thereof, attached to a substrate under conditions
that allow binding of the filovirus-specific antibody to the
detection agent; and (b) detecting the filovirus-specific antibody
bound to the detection agent, wherein the detection of the
filovirus-specific antibody identifies that the subject is infected
with a filovirus.
[0074] In yet another aspect, the disclosure provides a method for
identifying whether a subject is infected with a filovirus,
comprising: determining whether an antibody to the filovirus is
present in a sample obtained from the subject, wherein the
determining includes: (a) contacting the sample with at least one
protein, or a fragment thereof, from the filovirus to which the
antibody can specifically bind; and (b) detecting specific binding
between the at least one protein or the fragment thereof and the
antibody, wherein the detection of specific binding identifies that
the subject is infected with a filovirus.
[0075] Similarly to the embodiments relating to the detection agent
discussed herein, in some embodiments of the aspects relating to
the above methods, the filovirus may be selected from the group
consisting of Marburg marburgvirus, Sudan ebolavirus, Zaire
ebolavirus, Reston ebolavirus, Bundibugyo ebolavirus, and Tai
Forest ebolavirus.
[0076] In some embodiments the at least one protein or fragment
thereof comprises nucleoprotein (NP), virion protein 40 (VP40),
glycoprotein (GP), virion protein (VP35), virion protein (VP30),
virion protein (VP24), RNA-dependent RNA polymerase (L), or any
combination thereof, from any one or more filoviruses. Any amino
acid sequence that provides for binding and recognition of a
filovirus specific antibody may be used in connection with the
method. In some embodiments, the amino acid sequence may exhibit
little to no cross-reactivity to filovirus specific antibodies that
are directed to a particular type of filovirus or a particular
filovirus protein. In certain embodiments the one or more amino
acid sequences of a filovirus protein comprises GP, or fragment
thereof. The GP or fragment thereof may comprise a mucin-like
domain fragment of GP (GP-mucin) or a GP ectodomain (GP.DELTA.TM).
While the methods comprise at least one amino acid sequence of a
filovirus protein, the methods may also comprise a plurality of
such amino acid sequences from a single filovirus protein, and/or a
plurality of filovirus proteins, and/or a plurality of filoviruses.
As illustrated in the non-limiting Examples, the methods may
include at least three, at least four, at least five or at least
six different amino acid sequences from filovirus proteins, or
fragments thereof from different filoviruses (e.g., NP, VP40, and
GP-mucin, GP-ectodomain, etc. or fragments thereof).
[0077] In some embodiments, the methods can include amino acid
sequences that are not identical to the protein sequence(s) from
which the sequences are derived. Some minor changes in the primary
amino acid sequence and/or post-translational modification and
processing of the sequence may be included as long as the sequence
change or modification does not interfere with the ability of the
filovirus-specific antibody to bind the sequence. In some
embodiments, the amino acid sequences have at least 90% sequence
identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%) to the filovirus protein from which the sequence(s) are
derived. In some embodiments, the amino acid sequences may comprise
an NP sequence having at least 90% sequence identity to the
sequence selected from the group consisting of SEQ ID NO:4 (Zaire
NP); SEQ ID NO: 10 (Sudan NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ
ID NO: 22 (Tai Forest NP); SEQ ID NO: 28 (Reston NP); and SEQ ID
NO: 34 (Marburg NP); a VP40 sequence having at least 90% sequence
identity to the sequence selected from the group consisting of SEQ
ID NO: 2 (Zaire VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14
(Bundibugyo VP40); SEQ ID NO: 20 (Tai Forest VP40); SEQ ID NO: 26
(Reston VP40); and SEQ ID NO: 32 (Marburg VP40); and/or a GP-mucin
domain having at least 90% sequence identity to the sequence
selected from the group consisting SEQ ID NO: 6 (Zaire GP-mucin);
SEQ ID NO: 12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo
GP-mucin); SEQ ID NO: 24 (Tai Forest GP-mucin); SEQ ID NO: 30
(Reston GP-mucin); and SEQ ID NO: 36 (Marburg GP-mucin). In further
embodiments, the methods may comprise an NP sequence selected from
the group consisting of SEQ ID NO: 4 (Zaire NP); SEQ ID NO: 10
(Sudan NP); SEQ ID NO: 16 (Bundibugyo NP); SEQ ID NO: 22 (Tai
Forest NP); SEQ ID NO: 28 (Reston NP); and SEQ ID NO: 34(Marburg
NP); a VP40 selected from the list consisting of SEQ ID NO: 2
(Zaire VP40); SEQ ID NO: 8 (Sudan VP40); SEQ ID NO: 14 (Bundibugyo
VP40); SEQ ID 20 (Tai Forest VP40); SEQ ID NO: 26 (Reston VP40);
SEQ ID NO: 32 (Marburg VP40); and/or a GP-mucin domain selected
from the group consisting of SEQ ID NO: 6 (Zaire GP-mucin); SEQ ID
NO: 12 (Sudan GP-mucin); SEQ ID NO: 18 (Bundibugyo GP-mucin); SEQ
ID NO: 24 (Tai Forest GP-mucin); SEQ ID NO: 30 (Reston GP-mucin);
and SEQ ID NO: 36 (Marburg GP-mucin). In certain embodiments of the
methods, the methods comprise the detection agent as described
herein.
[0078] In some embodiments, the methods include incubation of the
sample with a detection agent at temperature and for a period of
time. While the time and temperature of the incubation, or
reaction, may vary it will suitably fall within a range that allows
for the specific binding of a filovirus specific antibody to an
amino acid that it can bind, or in some embodiments, bind
specifically. A further embodiment provides for temperatures and
times that are effective to facilitate binding of an antibody in
the sample to a filovirus protein or a fragment thereof, while
avoiding nonspecific interaction between the antibodies and a
filovirus protein or a fragment thereof.
[0079] In embodiments of the above methods, the sample or
biological sample may comprise any biologically-derived material
that may contain antibodies, or in which antibodies are typically
present. In some embodiments, the sample may be derived from a
mammal having a functioning or compromised an immune system. In
some embodiments, the biological samples may comprise tissue,
cells, or a biological fluid, such as blood (including serum, or
whole blood obtained from a finger prick), GCF, amniotic fluid,
BALF, salvia, tears, urine, lymphatic fluid, sputum, or
cerebrospinal fluid taken from a mammal. In other embodiments, the
biological sample may comprise cell cultures, cell lysates, or
cellular fluids. In particular embodiments, the sample may comprise
blood or serum.
[0080] In one embodiment of the aspects relating to methods, the
methods can be used to detect and determine the presence of a
filovirus specific antibody in a sample. In further embodiments, a
method may comprise determining the amount of a filovirus specific
antibody or a complex between a filovirus specific antibody and one
or more filovirus antigens, proteins, or fragments thereof. Thus,
the amount may be measured by determining the amount of the
antibodies and/or complexes in a sample. Detection may be performed
by any method and technique routinely used in the art such as, for
example, using an antibody which is detectably labeled, or which
can be subsequently labeled. A variety of formats can be employed
to determine whether a sample contains a filovirus specific
antibody. Immunoassay methods useful in the method detection can
include, but are not limited to, dot blotting, western blotting,
protein chips, immunoprecipitation (IP), competitive and
non-competitive protein binding assays, enzyme-linked immunosorbant
assays (ELISA), and others commonly used and widely-described in
scientific and patent literature, and many employed commercially.
One of skill in the art can readily adapt known protein/antibody
detection methods for use in determining whether samples contain a
biomarker (e.g., a filovirus specific antibody) and the relative
concentration in the sample. Further, the methods can include
processing of the sample to isolate a target (e.g., a filovirus
specific antibody or complex thereof) using known techniques
including, for example, those described in Harlow & Lane,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1988).
[0081] Detection antibodies can be used in the methods and kits
disclosed herein, including, for example, western blots or ELISA,
to detect the formation of complexes formed between one or more
filovirus-specific antibody and an amino acid sequence of a
filovirus protein or fragment thereof. In such uses, it is possible
to immobilize either the antibody or complexes on a solid support.
Supports or carriers include any support capable of binding an
antigen or an antibody, and are generally known in the art. Such
supports may include, for example glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and
modified celluloses, polyacrylamides, ceramics, semiconductors,
metal, and magnetite.
[0082] In one embodiment, the concentration of an antibody and/or
one or more filovirus proteins or fragments thereof is determined
in a biological sample obtained from a subject, including, for
example, a human patient. For example, the antibody and/or the
filovirus antigen can be isolated or purified from a sample
obtained from cells, serum, tissue, or an organ of the subject, as
discussed herein, and the amount thereof is determined. In some
embodiments, the filovirus antibody and/or a complex comprising the
filovirus antibody and an amino acid sequence of a filovirus
protein or fragment thereof complex can be prepared from cells,
tissue or organ samples by coimmunoprecipitation using an antibody
immunoreactive with an interacting protein member, a bifunctional
antibody that is immunoreactive with two or more interacting
protein members of the protein complex, or an antibody selectively
immunoreactive with the antibody and/or the complex. In some
embodiments, bifunctional antibodies or antibodies immunoreactive
with only free interacting filovirus antibodies are used,
individual filovirus antibodies not complexed with other proteins
may also be isolated along with the protein complex containing such
individual antibodies. The complexes, filovirus specific antibodies
and filovirus antigens may be separated from other proteins and
biological materials/molecules in samples using methods known in
the art, e.g., size-based separation methods such as gel
filtration, or by removing the complex from the sample using
another antibody having specific binding activity for the complex,
filovirus antibody, and/or filovirus protein. Additionally,
antibodies and proteins (and complexes between them) in a sample
can be separated in a gel such as polyacrylamide gel and
subsequently immunoblotted using an antibody immunoreactive with
the protein and/or complex.
[0083] Alternatively, the concentration can be determined in a
sample without separation, isolation or purification. For this
purpose, an antibody selectively immunoreactive with the
filovirus-specific antibody, filovirus antigen, and/or complex may
be used in an immunoassay. For example, immunocytochemical methods
can be used. Other antibody-based techniques are suitable and are
generally known in the art including, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA),
immunoradiometric assays (IRMA), fluorescent immunoassays, protein
A immunoassays, and immunoenzymatic assays (IEMA).
Methods of Manufacture
[0084] The disclosure also provides a method for making the
detection agent as described herein comprising: expressing one or
more recombinant polynucleotide sequences encoding an amino acid
sequence of a filovirus protein, or a fragment thereof in an
expression system; and fixing the encoded amino sequence of a
filovirus protein, or a fragment thereof, on a surface of the
substrate. The expression system includes a prokaryotic cell, a
eukaryotic cell, or in vitro translation, or any combination
thereof. The prokaryotic cell comprises E. coli. The eukaryotic
cell is selected from the group consisting of yeast, an insect
cell, and a mammalian cell.
[0085] In these embodiments, the methods further comprise the
general techniques and reagents that are known in the art and which
may find common use in preparing detection agents (e.g., protein
microarrays, protein-based microparticles, and/or protein-based
nanoparticles).
Kits
[0086] Another aspect relates to a kit including a detection agent
as disclosed herein, at least one reagent that can detect a
filovirus-specific antibody bound to the detection agent, and
instructions for use of the kit. A kit may be used for conducting
the diagnostic and screening methods described herein. Typically,
the kit should contain, in a carrier or compartmentalized
container, and additional reagents and buffers useful in any of the
above-described embodiments of the diagnosis method. The carrier
can be a container or support, in the form of, for example, bag,
box, tube, rack, and is optionally compartmentalized. The carrier
may define an enclosed confinement for safety purposes during
shipment and storage. In one embodiment, the kit includes an
antibody that is selectively immunoreactive with a complex
comprising a filovirus-specific antibody and an amino acid sequence
of a filovirus protein, or fragment thereof as described herein. In
some embodiments the kit includes an antibody that is selectively
immunoreactive with a filovirus-specific antibody (e.g., an
anti-mammal antibody, such as an anti-human antibody) that can
detect the presence of the filovirus-specific antibody bound to the
detection agent. The antibodies may be labeled with a detectable
marker such as radioactive isotopes, or enzymatic or fluorescence
markers. Alternatively, additional secondary antibodies such as
labeled anti-IgG and the like may be included for detection
purposes. Optionally, the kit can include one or more of proteins
sequences as a control or comparison purposes. Instructions for
using the kit or reagents contained therein are also included in
the kit.
[0087] The above aspects and embodiments are further illustrated in
the non-limiting Examples that follow. While the Examples may refer
to specific aspects of the disclosure, it will be appreciated that
the information is merely provided for purposes of illustration and
exemplification of the broader disclosure.
EXAMPLE 1
[0088] Cloning. Full-length genes for NP and VP40, and the GP
mucin-like domain fragment (GP-mucin) for six filovirus species:
Reston (REBOV), Bundibugyo (BEBOV), Zaire (ZEBOV), Sudan (SEBOV),
and Tai Forest ebolavirus (TAFV); and Marburg marburgvirus (MARV)
were cloned into pENTR.TM./TEV/D-TOPO.RTM. vector (Life
Technologies, Grand Island, N.Y.) and sequence-verified. The
nucleotide substitutions found in cloned sequence compared with the
reference sequence from GenBank are summarized in Table 1. All
entry vector clones were shuttled into destination E. coli
expression vectors via LR reaction (LR Clonase.RTM. II, Life
Technologies). Specifically, VP40 (Zaire VP40--SEQ ID NO: 1; Sudan
VP40--SEQ ID NO: 7; Bundibugyyo VP40--SEQ ID NO: 13; Tai Forest
VP40--SEQ ID NO: 19; Reston VP40--SEQ ID NO: 25; and Marburg VP
40--SEQ ID NO: 31) and GP-mucin (Zaire GP-mucin--SEQ ID NO: 5;
Sudan GP-mucin--SEQ ID NO: 11; Bundibugyo GP-mucin--SEQ ID NO: 17;
Tai Forest GP-mucin--SEQ ID NO: 23; Reston GP-mucin--SEQ ID NO: 29;
and Marburg GP-mucin--SEQ ID NO: 35) ORFs were shuttled into
pDESTHisMBP (Addgene plasmid 11085) containing an N-terminal HisMBP
tag, while all NP (Zaire NP--SEQ ID NO: 3; Sudan NP--SEQ ID NO: 9;
Bundibugyo NP--SEQ ID NO: 15; Tae Forest NP--SEQ ID NO: 21; Reston
NP--SEQ ID NO: 27; and Marburg NP--SEQ ID NO: 33) ORFs were
shuttled into pDEST17 (Life Technologies) containing an N-terminal
His tag.
TABLE-US-00001 TABLE 1 Summary of cloned filovirus sequences
compared to GenBank reference sequences Amino Acid GenBank
Nucleotide Species.sup.1 Gene Residues Sequence Substitutions.sup.3
Bundibugyo VP40 1-326 FJ217161.1 None ebolavirus NP 1-739
FJ217161.1 C1735T (silent) GP-mucin 313-465 FJ217161.1 C6973T
(silent), A7363G (silent) Tai Forest VP40 1-326 FJ217162.1 None
ebolavirus NP 1-739 FJ217162.1 None GP-mucin 313-465 FJ217162.1
None Reston VP40 1-331 AF522874.1 G4490A (silent), A5466G (Asn to
Asp) ebolavirus NP 1-739 AF522874.1 T2188C (silent) (Pennsylvania)
GP-mucin 314-466 AY769362. G7093A (silent) Sudan VP40 1-326
FJ968794.1 T4465C (silent) ebolavirus NP 1-738 AF173836.1 C2581T
(silent) (Boniface) GP-mucin 313-465 FJ968794.1 A7112G (silent)
Zaire VP40 1-326 AF499101.1 G4496A (silent), A4592G (silent),
ebolavirus T5204C (silent) (Mayinga) NP 1-739 AF086833.2 A491G (Ile
to Val) GP-mucin.sup.2 313-465 JQ352783.1 None Marburg VP40 1-303
DQ217792.1 None marburgvirus NP 1-695 DQ217792.1 None (Musoke)
GP-mucin 289-505 DQ217792.1 A6906T (silent) .sup.1Where available,
strain names are in parentheses. .sup.2Zaire ebolavirus GP-mucin
sequence is from the Kikwit strain. .sup.3The position of
nucleotide substitutions are based on GenBank sequence as
reference. Corresponding amino acid change is noted in parentheses.
Otherwise, the mutation is noted as silent.
[0089] Protein expression and purification. Proteins VP40 (Zaire
VP40--SEQ ID NO: 2; Sudan VP40: SEQ ID NO: 8; Bundibugyo VP40--SEQ
ID NO: 14; Tai Forest VP40--SEQ ID NO: 20; Reston VP40--SEQ ID NO:
26; and Marburg VP40--SEQ ID NO: 32), NP (Zaire NP--SEQ ID NO: 4;
Sudan NP--SEQ ID NO: 10; Bundibugyo NP--SEQ ID NO: 16; Tai Forest
NP--SEQ ID NO: 22; Reston NP--SEQ ID NO: 28; and Marburg NP--SEQ ID
NO: 34), GP-mucin (Zaire GP-mucin--SEQ ID NO: 6; Sudan
GP-mucin--SEQ ID NO: 12; Bundibugyo GP-mucin--SEQ ID NO: 18; Tai
Forest GP-mucin--SEQ ID NO: 24; Reston GP-mucin--SEQ ID NO: 30; and
Marburg GP-mucin--SEQ ID NO: 36) were expressed in either
BL21-AI.TM. cells (Life Technologies) or Rosetta.TM. 2(DE3) cells
(EMD Millipore, Billerica, Mass.). Expression for pDESTHisMBP
constructs was induced with 1 mM IPTG, while expression for pDEST17
18.degree. C. was induced lysed using with 0.2% L-arabinose.
Pelleted cells from overnight cultures grown at 18.degree. C. were
lysed using B-PER reagent (Thermo Scientific, Rockford, Ill.)
supplemented with 2.times. HaIt.TM. Protease and Phosphatase
Inhibitors Cocktail, EDTA-free (Thermo Scientific); 0.2 mg/mL
lysozyme; 50-100 U/mL DNasel (Thermo Scientific); and 2 mM PMSF.
Lysates were separated into supernatant and insoluble pellet
fractions by centrifugation, and induced protein expression was
confirmed through Western blotting or mass spectrometry and
Coomassie staining. HisMBP-tagged VP40s and GP-mucins were soluble
and present in the supernatant fraction. With the exception of
ZEBOV NP (Zaire-NP) (SEQ ID NO: 4), all His-tagged NPs were
insoluble and predominantly in the pellet fraction. Supernatant
containing expressed VP40s were loaded onto HisTrap.TM. HP columns
(GE Healthcare, Piscataway, N.J.) pre-equilibrated with 20 mM
sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 7.4. VP40
fractions were collected by applying an imidazole step elution. All
GP-mucins except MARV GP-mucin (Marburg-GP-mucin) were purified
using HisTrap.TM. HP columns. Binding and washing steps were
conducted with 25 mM HEPES, 0.5 M NaCl, 25 mM imidazole, pH 8, and
bound GP-mucins were eluted using an imidazole gradient.
Marburg-GP-mucin was purified using MBPTrap.TM. HP column
pre-equilibrated with 25 mM HEPES, 0.2 M NaCl, 1 mM EDTA, pH 7.4.
Bound protein was eluted using 25 mM HEPES, 0.2 M NaCl, 1 mM EDTA,
10 mM maltose, pH 7.4. NPs were purified through on-column
refolding on HisTrap.TM. HP columns. Briefly, NP pellets were
re-solubilized in 25 mM HEPES, 0.2 M NaCl, 25 mM imidazole, 1 mM
beta-mercaptoethanol, 6 M guanidine hydrochloride, pH 8. Proteins
were bound to columns under denaturing conditions and refolded
using a 6 to 0 M urea gradient over a 30 column volume range.
Refolded proteins were eluted using an imidazole gradient. Although
Zaire-NP was found in the supernatant, the protein did not appear
to bind to the HisTrap.TM. column under the conditions used for
VP40 purification. This may have been due to a hidden His tag, and
thus, guanidine hydrochloride was added directly to Zaire-NP
supernatant to a final concentration of 6 M in order to expose the
His tag. Denatured Zaire-NP was processed in a similar manner as
the other re-solubilized NPs. Purity and concentration of collected
fractions were measured by Agilent Protein 230 kit (Agilent
Technologies). All purified proteins were stored at -20.degree. C.
in their respective elution buffers with glycerol added to a final
concentration of 25%.
[0090] Microarray printing. The purified recombinant proteins were
spotted (FIG. 4A-B) on nitrocellulose-coated FAST.RTM. slides
(KeraFAST, Boston, Mass.), using a contactless inkjet microarray
printer (ArrayJet, Edinburgh, Scotland). The microarray included a
total of 34 proteins: i) E. coli-expressed filovirus antigens; ii)
insect cell-expressed ZEBOV and SEBOV and Marburg virus Angola) GP
ectodomain (.DELTA.TM) (IBT Bioservices, Gaithersburg, Md.); iii)
mammalian cell-expressed Marburg virus (Musoke) GP .DELTA.TM (IBT
Bioservices); iv) human, monkey, mouse, rabbit, and goat IgG
(Rockland Immunochemicals, Gilbertsville, Pa.); v) human, monkey,
and rabbit IgM (Rockland Immunochemicals; vi) HisMBP (ProteinOne,
Rockville, Md.); vii) dengue virus serotype 2 (dengue2) and 3
(dengue3) nonstructural protein 1 (NS1); and viii) BSA (Thermo
Scientific). The purified dengue virus proteins were previously
described (36). Briefly, the proteins were expressed with a HisMBP
tag in E. coli and purified via immobilized metal affinity
chromatography. Each protein was printed in triplicates. All
purified proteins were diluted to 200 ng/uL in printing buffer (25
mM HEPES, 0.5 M NaCl, 25% glycerol, 1 mM DTT, pH 8). Alexa
Fluor.RTM. 647-conjugated streptavidin (Life Technologies) was
diluted 1:50 in printing buffer and included in the microarray as
reference markers. Buffer served as empty placeholders on the
microarray. Printed slides were desiccated overnight under vacuum
and stored at -20.degree. C.
[0091] Microarray processing. All microarray processing steps were
performed under 21.degree. C. conditions, and each antibody or
serum sample was processed in duplicate microarrays. Printed
microarrays were incubated for 1 hour in 1.times. Biacore Flexchip
blocking buffer (GE Healthcare) with 2% normal goat serum (Vector
Laboratories, Burlingame, Calif.) or 2% normal rabbit serum.
Microarrays were washed three (3) times at five (5) minutes each
with wash buffer (1.times. TBS, 0.2% Tween 20, 3% BSA) which was
used in all subsequent wash steps. Microarrays were incubated with
primary antibody diluted 1:1000 or serum sample diluted 1:150 in
probe buffer (1.times. TBS, 0.1% Tween 20, 3% BSA). After 1 hour
incubation in primary antibody or sera, microarrays were washed and
incubated 1 hour with Alexa Fluor.RTM. 647-conjugated secondary
antibodies diluted 1:2000 in probe buffer. Microarrays were washed,
and then rinsed with water before analysis.
[0092] Vaccinations and infections. Rhesus macaque sera were
obtained from two separate vaccine studies for ZEBOV and MARV. The
vaccine trials were similar in design and procedure to a study
previously described by Warfield et al. (37). Briefly, for the
ZEBOV study, five animals were vaccinated with ZEBOV virus-like
particles (VLP) and MARV VLP. The vaccinated animals were
subsequently challenged with ZEBOV. Three sera were collected for
each animal: naive, post-immunization, and post-challenge.
TheMarburg virus study was conducted in a similar manner, except
that the animals were vaccinated with MARV VLP and challenged with
MARV.
[0093] Antibodies. Rabbit polyclonal anti-ZEBOV NP (anti-Zaire-NP,
0301-012), mouse monoclonal anti-SEBOV GP (anti-Sudan-GP,
0202-029), mouse monoclonal anti-SEBOV VP40 (anti-Sudan-VP40,
0202-018), mouse monoclonal anti-ZEBOV GP (anti-Zaire-GP,
0201-020), rabbit polyclonal anti-ZEBOV VP40 (anti-Zaire-VP40,
0301-010), mouse monoclonal anti-Marburg virus (Musoke) GP
(anti-Marburg-GP, 0203-023), and mouse monoclonal anti-Marburg
virus (Musoke) VP40 (anti-Marburg-VP40, 0203-012) antibodies were
purchased from IBT Bioservices. Alexa Fluor.RTM. 647-conjugated
goat anti-mouse IgG (A21237) and goat anti-rabbit IgG (A21244)
antibodies were purchased from Life Technologies. Alexa Fluor.RTM.
647-conjugated rabbit anti-monkey IgG (bs-00335R-A647) and rabbit
anti-monkey IgM (bs-0336R-A647) antibodies were purchased from
Bioss (Woburn, Mass.).
[0094] Data acquisition and analysis. Processed slides were scanned
at 635 nm wavelength using GenePix.RTM. 4400A (Molecular Devices,
Sunnyvale, Calif.). Acquired images were analyzed with GenePix Pro
7 software. Any defective or missing spots were removed from
further analysis. Median fluorescence intensity for each microarray
spot was corrected through local background subtraction on GenePix
Pro 7. Subsequent analysis was done in Microsoft Excel and R. The
resulting background-corrected fluorescence intensities were
averaged across replicate spots and quantile-normalized for each
serum group (naive, immunized, and challenged). A paired t-test was
conducted to compare each antigen-antibody signals for naive versus
immunized, naive versus challenged, and immunized versus challenged
sera.
RESULTS
[0095] Filovirus protein microarray. Taking into consideration the
complexity of the viral proteome and previous data suggesting
potential targets of host antibody responses (14-16, 38), we
developed a microarray comprised of a minimal set of proteins
representative of all Marburg and Ebola virus species. The VP40 and
NP for Reston, Bundibugyo, Zaire, Sudan, and Tai Forest ebolovirus
and Marburg marburgvirus MARV were expressed as full-length
recombinant proteins in E. coli. Initially, we prepared GP
ectodomains (.DELTA.TM) constructs from all filovirus species for
expression in E. coli. However, because the GP .DELTA.TM proteins
were not all stable in solution, the coding sequences were
truncated and expressed as more stable, GP mucin-like domain
fragments (Table 1), with HisMBP fusion tags (amino-termini). The
final GP protein design was supported by data from previous reports
suggesting that antibody responses to ZEBOV were directed at least
in part against the GP mucin-like domain (39-42).
[0096] The recombinant filovirus antigens purified from E. coli,
along with control proteins, were printed in 120-130 micron
diameter spots in a 12.times.12 format (FIG. 4) on slides covered
with a thin layer of nitrocellulose. Additionally, GP .DELTA.TM
produced in eukaryotic host cells were included in the microarray
for comparison with the E. coli-produced GP-mucins. IgGs (monkey,
human, rabbit goat, and mouse), IgMs (human, monkey, and rabbit),
HisMBP, BSA, and dengue virus proteins served as controls. For
quality control purposes and to validate our assay design, printed
microarrays were probed with anti-His antibody as well as a panel
of purified filovirus antibodies. Probing with anti-His antibody
showed that all His-tagged proteins were successfully spotted and
adsorbed onto the nitrocellulose-coated microarray slides (data not
shown). Anti-Marburg-VP40, anti-Marburg-GP, anti-Sudan-VP40,
anti-Sudan-GP, anti-Zaire-VP40, anti-Zaire-NP, and anti-Zaire-GP
were bound by their target antigens with a high degree of
specificity (FIG. 1A, B, C). Minor cross-reactivity between
REBOV-VP40 and Sudan-VP40, and between BEBOV-VP40 and ZEBOV-VP40
were observed when microarrays were probed with anti-SEBOV-VP40 and
anti-ZEBOV-VP40, respectively (FIG. 1 B, C). Combined, data from
these control antibodies indicate that the filovirus microarrays
performed correctly under idealized test conditions.
[0097] Analysis of sera from ZEBOV and MARV challenge studies. Sera
from two separate animal studies were analyzed using our
microarrays. In the ZEBOV study, rhesus macaques were vaccinated
with a mixture of trivalent (GP, NP, and VP40) virus-like particles
(VLP) for MARV and F and subsequently challenged with ZEBOV. In the
Marburg virus study, rhesus macaques were vaccinated with trivalent
(GP, NP, and VP40) VLP for MARV and subsequently challenged with
MARV. All vaccinated animals in the Zaire and Marburg studies
survived the viral challenge. After applying the serum samples to
the filovirus microarray, bound IgG were detected using
fluorescently-labeled secondary antibodies (FIGS. 5 and 6).
[0098] For the ZEBOV study, comparison between sera from naive and
immunized animals showed significant increases (p<0.05) in IgG
against all vaccine antigens except for MARV NP (Marburg-NP) (FIG.
2A). Cross-reactive IgG against BEBOV, TAFV, REBOV, and SEBOV VP40,
and BEBOV and TAFV NP were induced through vaccination (FIG. 2A).
After animals were challenged, IgG signals against all Ebola virus
NPs and VP40s and ZEBOV GP-mucin had significant increases
(p<0.005) in challenged sera compared to immunized sera (FIG.
2A). For the Marburg virus study, the microarrays detected
significant increases (p<0.05) in IgG against Marburg-NP,
-GP-mucin, and -VP40 in immunized sera compared to naive sera (FIG.
2B). Cross-reactive IgG against all Ebola virus VP40s were detected
in the immunized sera (FIG. 2B). We observed a cross-reactive
signal against Zaire-GP-mucin which was statistically significant
(p<0.05) comparing naive and immunized sera but not between
naive and challenged sera (FIG. 2B). Comparison between naive and
challenged sera showed significant increases (p<0.05) in IgG
responses for Marburg-NP and -GP-mucin (FIG. 2B). However, the
increase in IgG against Marburg-VP40 was not statistically
significant (FIG. 2B). The results from analysis of rhesus sera
suggested that the microarray enabled detection of anti-GP
antibodies in a species-specific manner. Further, the anti-GP
antibodies were detected with minimal cross-reactivity towards
other species for the case of sera from the ZEBOV and MARV
infections (FIG. 2C). We also examined IgM responses with sera from
both animal studies, and representative data are provided in
Supplementary FIG. 4. Overall, minor IgM signals were detected
against ZEBOV and MARV antigens using these convalescent sera. The
preliminary results indicate that the filovirus microarray may be
used for IgM detection. Analysis of sera collected from time points
closer to vaccination and viral challenge will confirm the utility
of measuring IgM responses by protein microarray.
[0099] Comparison between E. coli and eukaryotic cell-expressed GP.
Both the GP-mucins produced in E. coli and GP .DELTA.TMs produced
in eukaryotic cells (insect or mammalian) were included in the
printed microarray. Examining sera from the ZEBOV (FIG. 3A) and
MARV (FIG. 3B) studies, we confirmed that the mucin domain was
sufficient for capturing IgG responses to filoviruses. We observed
slightly higher IgG signals from the Zaire GP-mucin compared to
Zaire-GP .DELTA.TM with sera from ZEBOV challenged animals (FIG.
3A, B), whereas antibody recognition of the Marburg GP-mucin was
comparable to the GP .DELTA.TM Marburg (both Angola and Musoke) for
sera obtained from animals challenged with MARV. Based on these
microarray results, we concluded that the E. coli-produced GP-mucin
resulted in similar species-specificity as the eukaryotic
cell-expressed GP .DELTA.TMs.
DISCUSSION
[0100] As the above example and data show, the compositions and
methods disclosed herein, as demonstrated through one protein
microarray embodiment provide for a platform that can identify and
examine the antibody responses of mammals (e.g., rhesus macaques)
to infection and vaccination (e.g., various species of Ebola and
Marburg viruses). The illustrative florescence-based readout for
the microarray shows that the assay and methods are highly
sensitive, and only requires a minimal volume (1-2 microliters) of
sample in order to provide a complete evaluation. While any number
of amino acid sequences from one or more filovirus proteins may be
used in connection with the detection agent and methods, the NP and
GP antigens were very sensitive and could distinguish sera from
ZEBOV in comparison to Marburg virus infection. The results from
the Marburg virus study sera (Marburg-VP40) showed that some amino
acid sequences can be associated with an amount of cross-reactivity
to the VP40 antibody response against all Ebola viruses. Similarly,
the results shown herein also were able to identify a general
antibody cross-reactivity among Ebola virus NP and VP40 proteins,
which is similar to results from previously reported ELISA studies
(43-45). The data also identified that under these assay
conditions, GP exhibited the highest level of antibody specificity.
Further, and supporting the relevance of the GP-mucin domain as a
serological marker of infection, E. coli-expressed GP-mucins for
Zaire and Marburg filoviruses displayed similar species-specific
antibody recognition as the multi-domain GPs (.DELTA.TM) that were
produced from eukaryotic cells, based on assay results from the
ZEBOV and MARV studies. The microarray assay detected increases in
IgG responses to specific filovirus antigens resulting from
vaccination or viral challenge, and the relative levels of other
antibody isotypes (IgM) could also be measured. We further noted
that active infection stimulated a significant boost in immune
responses primed by vaccination, as specific IgG levels in
VLP-vaccinated macaques increased in response to aerosol challenges
from either ZEBOV or MARV. The significant increase in ZEBOV and
MARV-specific IgG following viral challenge, as measured by the
protein microarray, shows that VLP vaccinations did not induce
sterilizing immunity in the animals.
[0101] The results corroborate previously reported studies
concerning antibody recognition of filovirus antigens. Antibody
responses against NP and GP are detected in human patients by ELISA
(14-16) and Western blots (46). Other reports have observed
antibodies that recognize GP, NP, and VP40 in sera from a SEBOV
(Gulu) outbreak in 2000-2001(38). However, these previous ELISA
studies examined only select antigens from a single filovirus
species or a single antigen from multiple filoviruses, whereas the
microarray format supports a highly multiplex analysis of sera. By
providing these Examples which demonstrate that more than one
species of filovirus (e.g., two species of virus) can be examined
and identified using the techniques and compositions disclosed
herein, the disclosure provides for detection agents and methods
(e.g., protein microarray) useful for multiplexed study of
serological responses to most filovirus strains. The disclosure
expands the capabilities of any previously described methods and
compositions and facilitates diagnosis and serological surveillance
of infections caused by multiple species of the highly infectious
filoviruses. Further, providing for detection agents and methods
that can be adapted into a low-cost, point-of-care assay greatly
extends the utility of the technology, (e.g., relative to the prior
methods and techniques requiring full laboratory facilities).
[0102] Management and patient care for typical filoviral infections
provide significant challenges given the usually resource-poor
settings of outbreaks and the procedures that are required to
prevent spread of infections (47). Allaranga and coworkers proposed
that an active epidemiological surveillance system, including
surveillance of zoonotic infections, is vital for early detection
and effective response to filoviral hemorrhagic fever epidemics in
Africa (48). A recent report of hospital-based surveillance in
Ghana identifies the importance of distinguishing infections caused
by hepatitis viruses that produce symptoms that mimic viral
hemorrhagic fevers from the infrequent infections caused by
filoviruses (49). Further, the prevailing hypothesis concerning
outbreaks of filoviral hemorrhagic fevers is that indigenous human
populations occasionally make contact with animal reservoirs of
Ebola and Marburg viruses, resulting in rapid spread of disease
(Mbonye et al, 2013). Wildlife are often more severely affected
than humans, as demonstrated by a 89% drop in chimpanzees and 50%
decrease in gorilla populations as a result of one recorded Ebola
virus outbreak (50). Thus, the disclosure provides compositions and
assay methods that can be incorporated as a vital tool for such
epidemiological studies and for eventual diagnosis of infections,
including supporting serological surveillance of infections
occurring within domestic or wildlife animal populations.
EXAMPLE 2
[0103] This example illustrates the specificity of antibody
responses with sera collected from survivors of three separate
Ugandan outbreaks that were caused by Marburg marburgvirus (MARV)
in Kabale, Bundibugyo ebolavirus, (BDBV) in Bundibugyo, and Sudan
ebolavirus (SUDV) in the Gulu district. Control samples collected
from the same geographical regions as the disease outbreaks were
also included in the study. To measure antibody responses, we
assembled a protein microarray that displayed nucleoprotein (NP),
varion protein 40 (VP40), and glycoprotein (GP) antigens from
isolates representing the six species of filoviruses. Analysis of
the microarray data by hierarchical clustering revealed clear
positive signals from all infection samples, which were readily
distinguishable from negative controls. The amino acid sequences of
GP are most diverse among species, whereas NP sequences are highly
conserved. Consistent with protein similarities, NP was most
cross-reactive and exhibited the highest level of antibody
responses, while antibody responses to GP were the most specific.
Persistent antibody levels to GP, NP and VP40 were observed for
Gulu SUDV survivors 14 years after infection. Significant antibody
responses to autologous antigens were observed for all three
outbreak cohorts. The MARV survivors presented the lowest level of
antibody cross-reactivity with proteins from heterologous
filoviruses, while the SUDV survivors exhibited the highest
cross-reactivity with other filoviral proteins. Our results suggest
that survival from infection caused by one species of filovirus may
impart at least partial immunity to other outbreaks.
[0104] Methods:
[0105] Ebola and Marburg Survivor Sera
[0106] Our study included a total of 59 serum samples from patients
who survived infections caused by SUDV-Gulu, BDBV-Bundibugyo and
MARV-Kabale outbreaks along with controls from subjects living in
the same location who were not infected with the virus. The
survivors received uniform treatment after admission to a hospital.
Institutional approvals for the study were obtained from the Uganda
Virus Research Institute in Entebbe, Uganda, Ugandan National
Council for Science and Technology and United States Army Medical
Research Institute of Infectious Diseases (USAMRIID). A signed
consent form and a personal health questionnaire were obtained from
each subject. A serum sample from a human subject who was
vaccinated with a recombinant adenovirus serotype 5 (rAd5)
expressing EBOV and SUDV GP was also included in the study
(Ledgerwood, Costner et al. 2010).
[0107] Sequence and Phyologeny Analysis
[0108] Three multiple sequence alignments (MSAs) were generated for
the amino acid sequences of NP and GP mucin-like domains of
Ebolavirus and Marburgvirus strains, using CLUSTAL W2 (Larkin,
Blackshields et al. 2007). Each MSA had a different gap opening
penalty (5, 10, and 25), with Blosum62 as the protein weight matrix
and all other options left as default. T-Coffee Combine (Notredame,
Higgins et al. 2000, Di Tommaso, Moretti et al. 2011) was then used
to generate a single alignment that had the best agreement of all
three MSAs for each protein. The combined alignments of full-length
NP and GP mucin-like domain sequences were used to calculate
Shannon entropy per column of the aligned sequences as a measure of
amino acid variability and to generate percent identity matrices in
BioEdit Sequence Alignment Editor v7.1.3.0 (Hall 1999). To
eliminate poorly aligned positions and divergent regions in the
combined alignments, each alignment was filtered using Gblocks
(Castresana 2000, Talavera and Castresana 2007) with strict
settings (no gap positions within the final blocks, strict flanking
positions, and no small final blocks). Gblocks identified a 406
residue conserved region at the N-terminus of NP, which was used
for phylogenic reconstruction (BDBV, TAFV, RESTV, SUDV,
EBOV-residues 20-425; MARV-residues 2-407). Due to a high degree of
heterogeneity among individual residues, conserved regions within
GP mucin-like domain sequences could not be identified. For this
reason, an ungapped, highly variable region of 33 residues at the
N-terminal portion of the GP moiety was selected for use in
phylogenic reconstruction (BDBV, TAFV, RESTV-residues 2-34; EBOV
and MARV-residues 1-33). Phylogenic trees were generated using the
maximum likelihood method implemented in the PhyML program (v3.0
aLRT) (Guindon, Dufayard et al. 2010). The Blosum62 substitution
model was selected and 4 gamma-distributed rate categories to
account for rate heterogeneity across sites. The gamma shape
parameter was estimated directly from the data
(.alpha..sub.NP=0.654, .alpha..sub.GPmucin=15.371). Tree topology
and branch length were optimized for the starting tree with subtree
pruning and regrafting (SPR) selected for tree improvement.
Reliability for internal branches was assessed using a bootstrap
method with 1000 replicates. Comparison of phylogenetic trees was
completed using Compare2Trees software available online (Nye, Lio
et al. 2006).
[0109] Microarray Proteins
[0110] Recombinant proteins from filoviruses were cloned, and the
GP-mucin, NP and VP40 were expressed in E. coli, while the
GP.DELTA.TM proteins were produced in insect or mammalian
expression systems as previously described (Kamata, Natesan et al.
2014). The GP.DELTA.TM for BDBV, SUDV (Boniface), Zaire Ebola virus
(EBOV-Mayinga), Reston Ebola virus (RESTV-Reston), and MARV
(Angola) were produced in insect cells; while BDBV, RESTV
(Pennsylvania), Tai Forest Ebola virus (TAFV), SUDV (Boniface),
EBOV (Mayinga), and MARV (Musoke) GP.DELTA.TM were expressed in
mammalian cells. The Ebola GP-mucins were purified using HisTrap HP
columns and MARV GP-mucin was purified using MBPTrap HP column. The
NPs were purified by on-column refolding on HisTrap HP columns as
described previously (Kamata, Natesan et al. 2014). The purity and
concentrations of the proteins were determined by microfluidic
assays (Agilent Technologies, Santa Clara, Calif.). The dengue
virus proteins were previously described (Fernandez, Cisney et al.
2011).
[0111] Multiplexed Protein Microarray
[0112] The recombinant proteins were spotted (140 .mu.m diameter)
in a 10.times.36 microarray on FAST.RTM. slides (Kerafast, Boston,
Mass.) by using a Marathon inkjet microarrayer (ArrayJet,
Edinburgh, Scotland, UK). The array included a total of 41
proteins: i) E. coli-expressed filoviral antigens; ii)
Sf9-expressed GP.DELTA.TM from MARV, EBOV, SUDV, BDBV and RESTV
(IBT Bioservices); iii) mammalian cell-expressed GP.DELTA.TM from
from all six species of Filoviridae iv) human, monkey, mouse,
rabbit, and goat IgG (Rockland Immunochemicals, Gilbertsville,
Pa.); v) HisMBP (ProteinOne, Rockville, Md.); iv) human, monkey,
and rabbit IgM (Rockland Immunochemicals) and vi) dengue virus
serotype 2, 3 nonstructural protein 1 (NS1); and vi) BSA (Thermo
Fisher Scientific, Grand Island, N.Y.). The expression and
purification of the dengue virus proteins was previously described
(Fernandez, Cisney et al. 2011). Each protein in the microarray was
printed in quadruplicate. All purified proteins were diluted to 200
ng/.mu.L and prepared in a final concentration of 50% glycerol in
printing buffer consisting of 25 mM HEPES, 0.5 M NaCl, and 1 mM
dithiothreitol (DTT). Alexa647.RTM.-conjugated streptavidin (Life
Technologies) was diluted 1:50 in 1.times. PBS with 50% glycerol
and included in fixed positions within the array as a spatial
reference marker. The recombinant proteins were physically
characterized to confirm correct molecular weight and purity
(70-95%; data not shown). Quality control of printed microarrays
was confirmed with specific antibodies against poly-His tags,
Marburg VP40, Marburg GP, Sudan VP40, Sudan GP, anti-Zaire VP40,
Zaire NP and Zaire GP, as described previously (Kamata and Natesan,
2014). The printed and dried microarrays were stored under vacuum
(-20.degree. C.).
[0113] Analysis of Antibody Interactions
[0114] A Tecan HS Pro400 (Tecan US, Morrisville, N.C.)
hybridization station was used for most of the microarray
processing steps, and all manipulations were performed at
22.degree. C. The printed microarray slides were incubated for 1
hour in blocking buffer (1.times. Biacore Flexchip, GE Healthcare)
with 2% normal goat serum (Vector Laboratories). The microarray
slides were washed (3 times; 5 minute each) with a buffer (1.times.
TBS, 0.2% Tween 20, 3% BSA) that was used in all subsequent wash
steps. Human sera diluted 1:150 in probe buffer (1.times. TBS) were
incubated (1 hour) on the microarray surface. The slides were
washed and incubated for 1 hour with Alexa.RTM.647-conjugated
secondary antibodies diluted 1:2000 in probe buffer. The slides
were rinsed with water and dried before acquiring data.
[0115] Data Acquisition and Analysis
[0116] Processed slides were scanned at 635 nm wavelength using a
GenePix.RTM. 4400A (Molecular Devices, Sunnyvale, Calif.), with PMT
gain set to 400 and laser power set to 10%. Acquired images were
analyzed using GenPix Pro 7 software (Molecular Devices).
Background median fluorescence intensity was subtracted from median
fluorescence intensity of each spot, and the resulting
background-corrected fluorescence intensity was averaged across the
quadruplicates. The calculated mean fluorescence was used for
further analysis. The data were quantile normalized using the
preprocess core package of R. Background correction, standard
Z-score normalization to compare each signal to that of all
signals, and M--statistics were with performed with ProtoArray
Prospector software (Life Technologies), as described previously
(Keasey, Schmid et al. 2009). Group comparisons between control and
survivors were performed with thresholds of normalized signals of
at least 500 relative florescence units (RFUs), and a minimal
signal difference of 200 RFU between two groups. Heat maps of
normalized and log2-transformed values were created using GENE-E
(Broad Institute, Cambridge, Mass.). Hierarchical clustering by
average linkage Euclidean distance was used to examine overall
patterns of antibody interactions.
[0117] Results
[0118] Amino Acid Sequence Diversity of Filovirus Proteins
[0119] To examine human antibody responses to filovirus infections
we first considered the selection of protein probes to include in
our analysis. We performed a phylogenic analysis of filoviral
proteins (FIG. 8) to identify highly conserved probes as well as
proteins that may provide an antibody response signature that was
unique to each species of virus. As demonstrated in FIG. 8, NP is
highly conserved among ebolaviruses (>60% sequence identity),
while NP from MARV shares only 30% sequence identity with
ebolavirus species (FIG. 8c). Approximately 400 amino acids of the
N-terminal portion of NP that showed a higher degree of similarity
among ebolavirus strains and MARV (Keasey, unpublished data;
(Sanchez, Kiley et al. 1992), was selected for phylogenic inference
(FIG. 8a). The small shape parameter value (.alpha..sub.NP=0.654)
of the gamma distribution used for construction of the dendrogram
based on NP sequences indicated that there was a relatively large
amount of rate variation, with many sites evolving very slowly and
select sites evolving at a high rate (Lio and Goldman 1998). Thirty
percent of NP residues (216 residues out of 739 total) were
completely conserved among the six strains examined, with an
average variability/residue=0.638 (data not shown), based on
Shannon entropy calculations per residue of the NP MSA. In
contrast, the mucin-like domain of GP exhibited minimal sequence
identity among all filovirus strains examined (sequence
identity=5-26%, 8). Phylogenic inference was based on only a 33
residue region at the N-terminus of the domain (FIG. 8b), due to
the fact that this was the only ungapped portion of the multiple
sequence alignment. The shape parameter of the gamma distribution
(.alpha..sub.GPmucin=15.371) for GP-mucin sequences was much larger
than that of NP sequences, indicating that most sights have roughly
similar rates of substitution (Lio and Goldman 1998). No residues
within the mucin-like domain were completely conserved, and the
average variability per residue was 50% greater than that of NP
sequences (H/res=1.07). Further, the MARV GP mucin-like domain
sequence is 225 residues in length versus 153 residues for
ebolavirus strains.
[0120] We measured topological features among filovirus strains
based on amino acid sequences of NP and GP-mucin (Compare2Trees
software tool), (Nye, Lio et al. 2006). Comparison of phylogenic
trees (FIG. 8 a, b) reveals a similar topology for NP and GP-mucin,
despite extensive sequence diversity among individual proteins. The
overall similarity between the two trees was found to be 77.8%,
with BDBV, TAFV, EBOV edges being 100% conserved between trees, and
SUDV, RESTV, and MARV edges exhibiting 66.7% similarity. The long
branch length of MARV separated this lineage of filoviruses from
all ebolavirus species.
[0121] Human Antibody Reponses to Filoviral Proteins
[0122] The study involved a total of 37 survivors from the 2000
SUDV-Gulu outbreak, 20 samples from 2007 BDBV-Bundibugyo outbreak,
and 2 samples from MARV-Kabale outbreak (Table 2). The sera samples
were collected from a year after outbreak for MARV-Kabale, seven
years later for BDBV-Bundibugyo, and twelve to fourteen years after
for SUDV-Gulu. Sera collected from non-infected individuals living
in the same geographical region of each outbreak, normal healthy
volunteers from the United States, and serum from a subject
vaccinated with a replication defective rAd5 vaccine expressing GP
antigens in a 1:1 ratio from EBOV of Zaire strain and SUDV of Gulu
strain (Ledgerwood, Costner et al. 2010) were included as controls.
To examine antibody interactions, dilutions of each serum were
incubated on the surface of the protein microarray, which included
eleven GP.DELTA.TM proteins produced by eukaryotic cell expression,
eighteen proteins (GP-mucin, NP and VP40 from six species)
expressed in E. coli (Table 3).
TABLE-US-00002 TABLE 2 Summary of sample used in the study Year of
Year of No. of No. of Location Species Outbreak Collection Samples
Controls Gulu SUDV 2000 2012/2014 37 4 (Uganda) Bundibugyo BDBV
2007 2014 20 3 (Uganda) Kabale MARV 2012 2013 2 -- (Uganda)
USE-NIAID EBOV -- -- 1 5 rAd5 vector SUDV vaccine (GP)
[0123] A heat map showing the analysis of all serological immune
responses to the filovirus proteome is illustrated in FIG. 9.
Hierarchical clustering (unsupervised) of the data by Euclidean
distance indicated that the human sera was organized into two major
clusters corresponding to infected and uninfected (negative)
control groups (FIG. 9). Within the negative control group, the
non-African, BDBV negative controls, and SUDV negative samples
clustered separately (FIG. 9). The non-African control sera
exhibited no antibody interactions with the filoviral proteins,
while sera collected in Uganda showed some but not significant
binding to filoviral proteins. The MARV, BDBV and SUDV convalescent
samples clustered as distinct groups within the infected sample
group (FIG. 9), indicating that the IgG signals from our
microarrays can be used to distinguish between MARV, BDBV and
SUDV-infected sera. The convalescent samples from all MARV, BDBV,
and SUDV showed strong reactivity towards autologous antigens (FIG.
10). The MARV group showed significant increases against autologous
GP-mucin (p<0.001), GP.DELTA.TM (insect, p<0.001), NP
(p<0.001) and VP40 (p<0.001) when compared to negative
controls. Significant reactivity was seen for BDBV sera samples
against BDBV antigens GP-Mucin (p<0.05), GP.DELTA.TM (insect,
p<0.05), NP (p>0.05) and VP40 (p<0.05). Similarly, the
SUDV sera samples showed significant increases in antibody binding
to SUDV GP.DELTA.TM (insect, p<0.05) and NP (p<0.005).
However, the increase against SUDV VP40 was not statistically
significant (Table 2). For the case of the rAd5-vaccinated
individual, a robust antibody response was observed against EBOV
GP.DELTA.TM (mammalian) but not against SUDV GP (Supplementary
Figure). The recombinant rAd5 vaccine expressed EBOV and SUDV GP
proteins, and most vaccinees produced antibodies against both
proteins, as detected by ELISA (Ledgerwood, Costner et al.
2010).
[0124] We examined the cross-reactivity of the Ebola and Marburg
convalescent sera against heterologous antigens (FIG. 11).
Comparing all survivor sera, antibodies from the MARV infection
cases were the least cross reactive with ebolavirus GP.DELTA.TM
antigens, while only the BDBV-GP mucin interacted significantly
(p<0.005) with MARV convalescent serum antibodies. The MARV sera
did cross-react with other heterologous antigens, but to a much
lower extent than BDBV and SUDV survivors. The SUDV sera exhibited
significant levels of antibodies against RESTV GP.DELTA.TM (insect,
p<0.05), RESTV NP (p<0.05), and TAFV NP (p<0.05). The BDBV
presented the highest cross reactivity among filovirus antigens.
Significant antibody binding was observed with EBOV NP (p<0.05),
EBOV VP40 (p<0.05), SUDV NP (p<0.05), TAFV VP40 (p<0.05),
RESTV GP.DELTA.TM (insect, p<0.05), and RESTV NP (p<0.05).
Among the three antigens, the NP showed the highest levels of
antibody cross-reactivity, followed by VP40, and GP proteins.
TABLE-US-00003 TABLE 3 Summary of recombinant proteins used in the
microarray Species Proteins MARV GP-mucin, GP.DELTA.TM (insect),
GP.DELTA.TM (mammalian), NP, VP40 EBOV GP-mucin, GP.DELTA.TM
(insect), GP.DELTA.TM (mammalian), NP, VP40 BDBV GP-mucin,
GP.DELTA.TM (insect), GP.DELTA.TM (mammalian), NP, VP40 SUDV
GP-mucin, GP.DELTA.TM (insect), GP.DELTA.TM (mammalian), NP, VP40
TAFV GP-mucin, GP.DELTA.TM (mammalian), NP, VP40 RESTV GP-mucin,
GP.DELTA.TM (insect), GP.DELTA.TM (mammalian), NP, VP40
[0125] Discussion
[0126] The data demonstrates that the compositions and methods
provided herein can identify antibody responses by human survivors
of Ebola and Marburg infection. Previous studies have reported that
most antibody production in hosts was directed mainly towards GP,
NP and VP40 proteins (Johnson, Wambui et al. 1986, Leroy, Baize et
al. 2000, Lee, Fusco et al. 2008). Hence, for this example, these
three proteins were used in a microarray application. The VP40 and
NP were expressed as full length recombinant proteins. The GP
proteins were produced in three different formats, namely GP-mucin,
insect cell expressed GP.DELTA.TM, and mammalian cell expressed
GP.DELTA.TM. The GP protein is extensively glycosylated (Lee, Fusco
et al. 2008). We included both multi-domain GP.DELTA.TM, expressed
as glycosylated proteins in insect or mammalian cell cultures, and
single domain GP-mucins that were produced as non-glycosylated
proteins in E. coli. The results presented here show that the
mucin-like domain contributes substantially to human antibody, as
polyclonal antibody recognition of the isolated GP mucin-like
domain was comparable to the GP.DELTA.TM recombinant protein (see
also, Kamata, Natesan et al. 2014).
[0127] Antibody responses to Ebola infections have been studied
using ELISA (Nakayama, Yokoyama et al. 2010) (Prehaud, Hellebrand
et al. 1998) (Saijo, Niikura et al. 2001) and Western blots (Leroy,
Baize et al. 2000). Nakayama et aL has used recombinant GP from
Ebola in ELISA to detect IgG and IgM from infected individuals.
Neutralizing humoral responses to Ebola proteins by human survivors
of SUDV (Gulu) infection by ELISA were also reported (Sobarzo,
Perelman et al. 2012) (Sobarzo, Groseth et al. 2013), and viral
cell cultures were used as antigens in an ELISA to study IgM and
IgG responses (Macneil, Reed et al. 2011). Antibody
cross-reactivity among Ebola and Marburg viruses is not
well-characterized at the protein level. The sera samples were
obtained from survivors of three different outbreaks that occurred
in Uganda (Gulu-2000, Bundibugyo-2007 and Kabale-2012). The time of
sera collection varied from one year (MARV), seven years (BDBV),
and fourteen years (SUDV) after the outbreak of the disease. Two
types of controls were used in this study. One group comprised of
sera from healthy controls collected from the same area (Uganda)
and the second group of sera collected from a completely different
geographical region (USA). The background antibody levels in
endemic areas were slightly elevated compared to sera obtained from
non-endemic areas, emphasizing the need to include appropriate
controls for correct interpretation of data.
[0128] The convalescent sera exhibited high levels of antibody
binding for all antigens from the same species of filovirus that
caused the infection (FIG. 9). Among the three antigens we tested,
antibody levels were highest for NP, followed by GP and VP40. The
NP protein is essential for replication of viral genome and
formation of the nucleocapsid. Each virion contains about 3200 NP
molecules (Bharat, Noda et al. 2012). The C-terminus region of the
NP protein is highly antigenic and many epitopes have been
identified in this region (Saijo, Niikura et al. 2001) (Changula,
Yoshida et al. 2013). Hence, it is not surprising that NP elicits
strong antibody responses in infected subjects, and our results
confirm this observation and corroborate other reports (Sobarzo,
Perelman et al. 2012). GP is the primary surface protein of the
virion and several experimental vaccines and antibody-based
therapeutics target GP. For the three different forms of
recombinant GP (GP-mucin, GP.DELTA.TM-insect,
GP.DELTA.TM-mammalian) used in our microarray, the level of
antibody binding was similar (FIG. 13 or data not shown) for all
six species except for insect cell produced BDBV GP.DELTA.TM, which
showed higher binding than GP-mucin and GP.DELTA.TM-mammalian.
Although the VP40 protein is the most abundant protein within the
mature virus, our results show that the antibody response to VP40
in humans is lower than that obtained with NP and GP proteins. It
is striking that 14 years after infection many individuals within
the SUDV survivor group retain antibodies against filoviral
proteins. Persistence of antifilovirus antibodies in long-term
survivors of infection was previously reported (Wauquier, Becquart
et al. 2009, Sobarzo, Ochayon et al. 2013).
[0129] Our study did not include human survivors from EBOV
infection from the current West African outbreak due to the
unavailability of samples. We did include in our study a serum
sample from a human subject vaccinated with chimpanzee rAd5 Ebola
vaccine developed by the National Institutes of Health (Ledgerwood,
Costner et al. 2010). The vaccine was bivalent, expressing both
EBOV and SUDV GP proteins. We found robust antibody response
against EBOV GP.DELTA.TM (mammalian) but not against SUDV GP
protein (FIG. 12). The absence and diminished response against GP
proteins may be due to the presence of pre-existing neutralizing
antibodies against the Ad5 vector. The capacity of pre-existing
vector-specific humoral responses to interfere with efficacy of
vaccines is well documented (Pine, Kublin et al. 2011, Ledgerwood,
DeZure et al. 2014). In our previous study (Kamata, Natesan et al.
2014) of rhesus macques that were vaccinated with EBOV VLP and
challenged with live EBOV, antibodies to GP presented the highest
level of specificity in contrast to the human survivor serum
antibodies, which showed appreciable amounts of cross-reactivity to
heterologous GP proteins. Combined, these results suggest that for
the GP antibody response, lower primate and humans may differ
depending on how each were exposed to filoviruses. Nakayama et al.
reported similar findings by comparing antibody responses to Ebola
by mice and humans (Nakayama, Yokoyama et al. 2010).
[0130] Cross-reactivity towards heterologous antigens can be
observed for all three groups of survivors (BDBV, SUDV and MARV).
This is expected since there is considerable amount of protein
sequence homology found between the five Ebola species (FIG. 8).
The MARV species show the least homology to other members of Ebola.
Our phylogenic analysis showed (FIG. 8) that NP is highly conserved
among the filoviral species, hence may have common epitiopes that
are present in all filovirus species. More than 30% of NP residues
are conserved among all six species (FIG. 8). The conserved region
of NP forms a condensed helix and may contain a structure that
plays a role in virus replication (Bharat, Noda et al. 2012). A
previous study (Changula, Yoshida et al. 2013) has identified
epitopes in this region that cross react to all or several members
of Filoviridae. However, the highly variable C terminal region of
NP contains the highest number of antigenic regions. This example
confirms that among the three antigens tested the NP showed the
most cross-reactivity, in agreement with previous reports (Sobarzo,
Groseth et al. 2013, Sobarzo, Ochayon et al. 2013, McElroy, Akondy
et al. 2015).
[0131] This example provides the first report describing a
multiplexed assay method and a multiplex protein microarray that
was used to identify and analyze antibody specificity and
cross-reactivity from human survivors of Ebola and Marburg. While
the data suggests a considerable amount of variability in human
antibody responses, it identifies GP as desirable targets for
neutralizing antibodies (as GP are responsible for virus entry into
cells). Nevertheless, the immune responses observed for VP40 and NP
can also serve as suitable diagnostic indicators and biomarkers of
infection. The disclosure thus provides for detection agents and
methods that can effectively identify filoviral-specific antibodies
in a sample, and further allows for the application of additional
proteins or lysates from filovirus and other new viruses that may
present outbreak concerns and issues of public health. Further, the
methods and compositions disclosed herein can distinguish Ebola
infections from diseases that mimic symptoms similar to that of
filoviral infections, and can be used as a tool for
seroepidemiological screening as well as for the diagnosis of
filoviral infections in mammals.
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Sequence CWU 1
1
361981DNAEbola virus 1atgaggcggg ttatattacc tactgctcct cctgaatata
tggaggccat ataccctgtc 60aggtcaaatt caacaattgc tagaggtggc aacagcaata
caggcttcct gacgccggag 120tcagtcaatg gggacactcc atcgaatcca
ctcaggccaa ttgccgatga caccatcgac 180catgccagcc acacaccagg
cagtgtgtca tcagcattca tccttgaagc tatggtgaat 240gtcatatcgg
gccccaaagt gctaatgaag caaattccaa tttggcttcc tctaggtgtc
300gctgatcaaa agacctacag ctttgactca actacggccg ccatcatgct
tgcttcatac 360actatcaccc atttcggcaa ggcaaccaat ccacttgtca
gagtcaatcg gctgggtcct 420ggaatcccgg atcatcccct caggctcctg
cgaattggaa accaggcttt cctccaggag 480ttcgttcttc cgccagtcca
actaccccag tatttcacct ttgatttgac agcactcaaa 540ctgatcaccc
aaccactgcc tgctgcaaca tggaccgatg acactccaac aggatcaaat
600ggagcgttgc gtccaggaat ttcatttcat ccaaaacttc gccccattct
tttacccaac 660aaaagtggga agaaggggaa cagtgccgat ctaacatctc
cggagaaaat ccaagcaata 720atgacctcac tccaggactt caagatcgtt
ccaattgatc caaccaaaaa tatcatggga 780atcgaagtgc cagaaactct
ggtccacaag ctgaccggta agaaggtgac ttctaaaaat 840ggacaaccaa
tcatccctgt tcttttgcca aagtacattg ggttggaccc ggtggctcca
900ggagacctca ccatggtaat cacacaggat tgtgacacgt gtcattctcc
tgcaagtctt 960ccagctgtga ttgagaagta a 9812326PRTEbola virus 2Met
Arg Arg Val Ile Leu Pro Thr Ala Pro Pro Glu Tyr Met Glu Ala 1 5 10
15 Ile Tyr Pro Val Arg Ser Asn Ser Thr Ile Ala Arg Gly Gly Asn Ser
20 25 30 Asn Thr Gly Phe Leu Thr Pro Glu Ser Val Asn Gly Asp Thr
Pro Ser 35 40 45 Asn Pro Leu Arg Pro Ile Ala Asp Asp Thr Ile Asp
His Ala Ser His 50 55 60 Thr Pro Gly Ser Val Ser Ser Ala Phe Ile
Leu Glu Ala Met Val Asn 65 70 75 80 Val Ile Ser Gly Pro Lys Val Leu
Met Lys Gln Ile Pro Ile Trp Leu 85 90 95 Pro Leu Gly Val Ala Asp
Gln Lys Thr Tyr Ser Phe Asp Ser Thr Thr 100 105 110 Ala Ala Ile Met
Leu Ala Ser Tyr Thr Ile Thr His Phe Gly Lys Ala 115 120 125 Thr Asn
Pro Leu Val Arg Val Asn Arg Leu Gly Pro Gly Ile Pro Asp 130 135 140
His Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu Gln Glu 145
150 155 160 Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe
Asp Leu 165 170 175 Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu Pro Ala
Ala Thr Trp Thr 180 185 190 Asp Asp Thr Pro Thr Gly Ser Asn Gly Ala
Leu Arg Pro Gly Ile Ser 195 200 205 Phe His Pro Lys Leu Arg Pro Ile
Leu Leu Pro Asn Lys Ser Gly Lys 210 215 220 Lys Gly Asn Ser Ala Asp
Leu Thr Ser Pro Glu Lys Ile Gln Ala Ile 225 230 235 240 Met Thr Ser
Leu Gln Asp Phe Lys Ile Val Pro Ile Asp Pro Thr Lys 245 250 255 Asn
Ile Met Gly Ile Glu Val Pro Glu Thr Leu Val His Lys Leu Thr 260 265
270 Gly Lys Lys Val Thr Ser Lys Asn Gly Gln Pro Ile Ile Pro Val Leu
275 280 285 Leu Pro Lys Tyr Ile Gly Leu Asp Pro Val Ala Pro Gly Asp
Leu Thr 290 295 300 Met Val Ile Thr Gln Asp Cys Asp Thr Cys His Ser
Pro Ala Ser Leu 305 310 315 320 Pro Ala Val Ile Glu Lys 325
32220DNAEbola virus 3atggattctc gtcctcagaa agtctggatg gcgccgagtc
tcactgaatc tgacatggat 60taccacaaga tcttgacagc aggtctgtcc gttcaacagg
ggattgttcg gcaaagagtc 120atcccagtgt atcaagtaaa caatcttgaa
gaaatttgcc aacttatcat acaggccttt 180gaagcaggtg ttgattttca
agagagtgcg gacagtttcc ttctcatgct ttgtcttcat 240catgcgtacc
agggagatta caaacttttc ttggaaagtg gcgcagtcaa gtatttggaa
300gggcacgggt tccgttttga agtcaagaag cgtgatggag tgaagcgcct
tgaggaattg 360ctgccagcag tatctagtgg aaaaaacatt aagagaacac
ttgctgccat gccggaagag 420gagacaactg aagctaatgc cggtcagttt
ctctcctttg caagtctatt ccttccgaaa 480ttggtagtag gagaaaaggc
ttgccttgag aaggttcaaa ggcaaattca agtacatgca 540gagcaaggac
tgatacaata tccaacagct tggcaatcag taggacacat gatggtgatt
600ttccgtttga tgcgaacaaa ttttctgatc aaatttctcc taatacacca
agggatgcac 660atggttgccg ggcatgatgc caacgatgct gtgatttcaa
attcagtggc tcaagctcgt 720ttttcaggct tattgattgt caaaacagta
cttgatcata tcctacaaaa gacagaacga 780ggagttcgtc tccatcctct
tgcaaggacc gccaaggtaa aaaatgaggt gaactccttt 840aaggctgcac
tcagctccct ggccaagcat ggagagtatg ctcctttcgc ccgacttttg
900aacctttctg gagtaaataa tcttgagcat ggtcttttcc ctcaactatc
ggcaattgca 960ctcggagtcg ccacagcaca cgggagtacc ctcgcaggag
taaatgttgg agaacagtat 1020caacaactca gagaggctgc cactgaggct
gagaagcaac tccaacaata tgcagagtct 1080cgcgaacttg accatcttgg
acttgatgat caggaaaaga aaattcttat gaacttccat 1140cagaaaaaga
acgaaatcag cttccagcaa acaaacgcta tggtaactct aagaaaagag
1200cgcctggcca agctgacaga agctatcact gctgcgtcac tgcccaaaac
aagtggacat 1260tacgatgatg atgacgacat tccctttcca ggacccatca
atgatgacga caatcctggc 1320catcaagatg atgatccgac tgactcacag
gatacgacca ttcccgatgt ggtggttgat 1380cccgatgatg gaagctacgg
cgaataccag agttactcgg aaaacggcat gaatgcacca 1440gatgacttgg
tcctattcga tctagacgag gacgacgagg acactaagcc agtgcctaat
1500agatcgacca agggtggaca acagaagaac agtcaaaagg gccagcatat
agagggcaga 1560cagacacaat ccaggccaat tcaaaatgtc ccaggccctc
acagaacaat ccaccacgcc 1620agtgcgccac tcacggacaa tgacagaaga
aatgaaccct ccggctcaac cagccctcgc 1680atgctgacac caattaacga
agaggcagac ccactggacg atgccgacga cgagacgtct 1740agccttccgc
ccttggagtc agatgatgaa gagcaggaca gggacggaac ttccaaccgc
1800acacccactg tcgccccacc ggctcccgta tacagagatc actctgaaaa
gaaagaactc 1860ccgcaagacg agcaacaaga tcaggaccac actcaagagg
ccaggaacca ggacagtgac 1920aacacccagt cagaacactc ttttgaggag
atgtatcgcc acattctaag atcacagggg 1980ccatttgatg ctgttttgta
ttatcatatg atgaaggatg agcctgtagt tttcagtacc 2040agtgatggca
aagagtacac gtatccagac tcccttgaag aggaatatcc accatggctc
2100actgaaaaag aggctatgaa tgaagagaat agatttgtta cattggatgg
tcaacaattt 2160tattggccgg tgatgaatca caagaataaa ttcatggcaa
tcctgcaaca tcatcagtga 22204739PRTEbola virus 4Met Asp Ser Arg Pro
Gln Lys Val Trp Met Ala Pro Ser Leu Thr Glu 1 5 10 15 Ser Asp Met
Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln 20 25 30 Gln
Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn 35 40
45 Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val
50 55 60 Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys
Leu His 65 70 75 80 His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu
Ser Gly Ala Val 85 90 95 Lys Tyr Leu Glu Gly His Gly Phe Arg Phe
Glu Val Lys Lys Arg Asp 100 105 110 Gly Val Lys Arg Leu Glu Glu Leu
Leu Pro Ala Val Ser Ser Gly Lys 115 120 125 Asn Ile Lys Arg Thr Leu
Ala Ala Met Pro Glu Glu Glu Thr Thr Glu 130 135 140 Ala Asn Ala Gly
Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys 145 150 155 160 Leu
Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile 165 170
175 Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln
180 185 190 Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr
Asn Phe 195 200 205 Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His
Met Val Ala Gly 210 215 220 His Asp Ala Asn Asp Ala Val Ile Ser Asn
Ser Val Ala Gln Ala Arg 225 230 235 240 Phe Ser Gly Leu Leu Ile Val
Lys Thr Val Leu Asp His Ile Leu Gln 245 250 255 Lys Thr Glu Arg Gly
Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys 260 265 270 Val Lys Asn
Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala 275 280 285 Lys
His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295
300 Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala
305 310 315 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly
Val Asn Val 325 330 335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala
Thr Glu Ala Glu Lys 340 345 350 Gln Leu Gln Gln Tyr Ala Glu Ser Arg
Glu Leu Asp His Leu Gly Leu 355 360 365 Asp Asp Gln Glu Lys Lys Ile
Leu Met Asn Phe His Gln Lys Lys Asn 370 375 380 Glu Ile Ser Phe Gln
Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu 385 390 395 400 Arg Leu
Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu Pro Lys 405 410 415
Thr Ser Gly His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro Gly Pro 420
425 430 Ile Asn Asp Asp Asp Asn Pro Gly His Gln Asp Asp Asp Pro Thr
Asp 435 440 445 Ser Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro
Asp Asp Gly 450 455 460 Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser Glu Asn
Gly Met Asn Ala Pro 465 470 475 480 Asp Asp Leu Val Leu Phe Asp Leu
Asp Glu Asp Asp Glu Asp Thr Lys 485 490 495 Pro Val Pro Asn Arg Ser
Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln 500 505 510 Lys Gly Gln His
Ile Glu Gly Arg Gln Thr Gln Ser Arg Pro Ile Gln 515 520 525 Asn Val
Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu 530 535 540
Thr Asp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg 545
550 555 560 Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp
Ala Asp 565 570 575 Asp Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp
Asp Glu Glu Gln 580 585 590 Asp Arg Asp Gly Thr Ser Asn Arg Thr Pro
Thr Val Ala Pro Pro Ala 595 600 605 Pro Val Tyr Arg Asp His Ser Glu
Lys Lys Glu Leu Pro Gln Asp Glu 610 615 620 Gln Gln Asp Gln Asp His
Thr Gln Glu Ala Arg Asn Gln Asp Ser Asp 625 630 635 640 Asn Thr Gln
Ser Glu His Ser Phe Glu Glu Met Tyr Arg His Ile Leu 645 650 655 Arg
Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys 660 665
670 Asp Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr
675 680 685 Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu
Lys Glu 690 695 700 Ala Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp
Gly Gln Gln Phe 705 710 715 720 Tyr Trp Pro Val Met Asn His Lys Asn
Lys Phe Met Ala Ile Leu Gln 725 730 735 His His Gln 5465DNAEbola
virus 5aacagagcca aaaacatcag tggtcagagt ccggcgcgaa cttcttccga
cccagggacc 60aacacaacaa ctgaagacca caaaatcatg gcttcagaaa attcctctgc
aatggttcaa 120gtgcacagtc aaggaaggga agctgcagtg tcgcatctga
caacccttgc cacaatctcc 180acgagtcctc aaccccccac aaccaaacca
ggtccggaca acagcaccca caatacaccc 240gtgtataaac ttgacatctc
tgaggcaact caagttgaac aacatcaccg cagaacagac 300aacgacagca
cagcctccga cactcccccc gccacgaccg cagccggacc cctaaaagca
360gagaacacca acacgagcaa gggtaccgac ctcctggacc ccgccaccac
aacaagtccc 420caaaaccaca gcgagaccgc tggcaacaac aacactcatt agtag
4656153PRTEbola virus 6Asn Arg Ala Lys Asn Ile Ser Gly Gln Ser Pro
Ala Arg Thr Ser Ser 1 5 10 15 Asp Pro Gly Thr Asn Thr Thr Thr Glu
Asp His Lys Ile Met Ala Ser 20 25 30 Glu Asn Ser Ser Ala Met Val
Gln Val His Ser Gln Gly Arg Glu Ala 35 40 45 Ala Val Ser His Leu
Thr Thr Leu Ala Thr Ile Ser Thr Ser Pro Gln 50 55 60 Pro Pro Thr
Thr Lys Pro Gly Pro Asp Asn Ser Thr His Asn Thr Pro 65 70 75 80 Val
Tyr Lys Leu Asp Ile Ser Glu Ala Thr Gln Val Glu Gln His His 85 90
95 Arg Arg Thr Asp Asn Asp Ser Thr Ala Ser Asp Thr Pro Pro Ala Thr
100 105 110 Thr Ala Ala Gly Pro Leu Lys Ala Glu Asn Thr Asn Thr Ser
Lys Gly 115 120 125 Thr Asp Leu Leu Asp Pro Ala Thr Thr Thr Ser Pro
Gln Asn His Ser 130 135 140 Glu Thr Ala Gly Asn Asn Asn Thr His 145
150 7981DNAEbola virus 7atgagaaggg tcactgtgcc gactgcacca cctgcatatg
ctgacattgg ctatcctatg 60agcatgcttc caatcaagtc aagcagggct gtaagtggaa
ttcaacagaa acaagaggtc 120cttcctggaa tggatacacc atcgaactct
atgagacctg ttgctgatga taacattgat 180cacacaagtc ataccccaaa
cggagtggcc tcagcattca tcttggaggc aactgtcaat 240gtgatctcgg
ggcccaaagt cctcatgaaa caaatcccta tttggttgcc actcggaatt
300gctgaccaaa aaacatacag ctttgactca acaacagcag caattatgct
cgcatcctac 360acgatcactc attttggaaa ggccaacaac cccctcgtca
gagtgaatcg acttggtcaa 420ggaataccgg atcacccact cagattgctc
aggatgggga accaggcttt ccttcaagag 480tttgtgctac caccagttca
actgccgcaa tatttcactt ttgatctgac tgcactcaaa 540ttagtgacac
agcctctccc tgctgcaaca tggacagatg agactccgag caacctttca
600ggagcactcc gtccagggct ctcatttcac ccgaaactga gacccgttct
acttccaggc 660aagacgggaa agaaagggca tgtttctgat ctgaccgccc
cagacaaaat ccagacaatt 720gtgaacctga tgcaagattt caagattgtg
ccaatcgacc cagccaagag catcattggg 780atcgaggttc cagaattgct
ggtccacaag ctcaccggga agaaaatgag tcagaagaac 840ggacagccta
taattcctgt cttactccca aaatacattg ggctagatcc aatctcgccc
900ggagacctaa ctatggtcat aacaccagat tatgatgatt gtcattcacc
cgccagttgc 960tcttatctca gtgaaaagtg a 9818326PRTEbola virus 8Met
Arg Arg Val Thr Val Pro Thr Ala Pro Pro Ala Tyr Ala Asp Ile 1 5 10
15 Gly Tyr Pro Met Ser Met Leu Pro Ile Lys Ser Ser Arg Ala Val Ser
20 25 30 Gly Ile Gln Gln Lys Gln Glu Val Leu Pro Gly Met Asp Thr
Pro Ser 35 40 45 Asn Ser Met Arg Pro Val Ala Asp Asp Asn Ile Asp
His Thr Ser His 50 55 60 Thr Pro Asn Gly Val Ala Ser Ala Phe Ile
Leu Glu Ala Thr Val Asn 65 70 75 80 Val Ile Ser Gly Pro Lys Val Leu
Met Lys Gln Ile Pro Ile Trp Leu 85 90 95 Pro Leu Gly Ile Ala Asp
Gln Lys Thr Tyr Ser Phe Asp Ser Thr Thr 100 105 110 Ala Ala Ile Met
Leu Ala Ser Tyr Thr Ile Thr His Phe Gly Lys Ala 115 120 125 Asn Asn
Pro Leu Val Arg Val Asn Arg Leu Gly Gln Gly Ile Pro Asp 130 135 140
His Pro Leu Arg Leu Leu Arg Met Gly Asn Gln Ala Phe Leu Gln Glu 145
150 155 160 Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe
Asp Leu 165 170 175 Thr Ala Leu Lys Leu Val Thr Gln Pro Leu Pro Ala
Ala Thr Trp Thr 180 185 190 Asp Glu Thr Pro Ser Asn Leu Ser Gly Ala
Leu Arg Pro Gly Leu Ser 195 200 205 Phe His Pro Lys Leu Arg Pro Val
Leu Leu Pro Gly Lys Thr Gly Lys 210 215 220 Lys Gly His Val Ser Asp
Leu Thr Ala Pro Asp Lys Ile Gln Thr Ile 225 230 235 240 Val Asn Leu
Met Gln Asp Phe Lys Ile Val Pro Ile Asp Pro Ala Lys 245 250 255 Ser
Ile Ile Gly Ile Glu Val Pro Glu Leu Leu Val His Lys Leu Thr 260 265
270 Gly Lys Lys Met Ser Gln Lys Asn Gly Gln Pro Ile Ile Pro Val Leu
275 280 285 Leu Pro Lys Tyr Ile Gly Leu Asp Pro Ile Ser Pro Gly Asp
Leu Thr 290 295 300 Met Val Ile Thr Pro Asp Tyr Asp Asp Cys His Ser
Pro Ala Ser Cys 305 310 315 320 Ser Tyr Leu Ser Glu Lys 325
92217DNAEbola virus 9atggataaac gggtgagagg ttcatgggcc ctgggaggac
aatctgaggt tgatcttgac 60taccacaaga
tattaacagc cgggctttca gtccaacagg ggattgtgcg acagagagtc
120atcccggtat atgtcgtgaa tgatcttgag ggtatttgtc aacatatcat
tcaggctttt 180gaagcaggtg tagatttcca ggataatgct gatagcttcc
ttttactttt atgtttacat 240catgcctacc aaggagatca taggctcttc
ctcaaaagtg atgcagttca atatttagag 300ggccatggct tcaggtttga
ggtccgagaa aaggagaatg tgcaccgtct ggatgaattg 360ttgcccaatg
ttaccggtgg aaaaaatctc aggagaacat tggctgctat gcccgaagag
420gagacaacgg aagctaatgc tggtcagttt ctatcctttg ccagtttgtt
tctacccaaa 480cttgtcgttg gggagaaagc gtgcctggaa aaagtacaaa
ggcaaattca ggtccatgca 540gaacaagggc tcattcaata tccaacttcc
tggcaatcag ttggacacat gatggtgatc 600ttccgtttga tgaggacaaa
ctttttaatc aagtttctac taatacatca agggatgcac 660atggttgcag
gtcatgatgc gaatgacaca gtaatatcta attctgttgc ccaggcaagg
720ttctctggtc ttctgattgt aaagactgtt ctggatcaca tcctacaaaa
aacagatctc 780ggagtacgac ttcatccact ggccaggaca gcaaaagtga
agaatgaggt cagttcattc 840aaggcggctc ttggttcact tgccaagcat
ggagaatatg ctccgtttgc acgtctcctt 900aatctttctg gagtcaacaa
cttggaacat gggctttatc cacaactttc agccatcgct 960ttgggtgttg
caactgccca cgggagtacg cttgctggtg tgaatgtagg ggagcaatat
1020cagcaactgc gtgaggctgc tactgaggct gaaaagcaac tccaacaata
tgctgaaaca 1080cgtgagttgg ataaccttgg gcttgatgaa caggagaaga
agattctcat gagcttccac 1140cagaagaaga atgagatcag cttccagcag
actaatgcaa tggtaacctt aaggaaagaa 1200cggctggcta aattgaccga
agccatcacg actgcatcga agatcaaggt tggagaccgt 1260tatcctgatg
acaatgatat tccatttccc gggccgatct atgatgacac tcaccccaat
1320ccctctgatg acaatcctga tgattcacgt gatacaacta ttccaggtgg
tgttgttgac 1380ccgtatgatg atgagagtaa taattatcct gactacgagg
attcggctga aggcaccaca 1440ggagatcttg atctcttcaa tttggacgac
gacgatgatg acagccgacc aggaccacca 1500gacagggggc agaacaagga
gagggcggcc cggacatatg gcctccaaga tccgaccttg 1560gacggagcga
aaaaggtgcc ggagttgacc ccaggttccc atcaaccagg caacctccac
1620atcaccaagt cgggttcaaa caccaaccaa ccacaaggca atatgtcatc
tactctccat 1680agtatgaccc ctatacagga agaatcagag cccgatgatc
aaaaagataa tgatgacgag 1740agtctcacat cccttgactc tgaaggtgac
gaagatggtg agagcatctc tgaggagaac 1800accccaactg tagctccacc
agcaccagtc tacaaagaca ctggagtaga cactaatcag 1860cagaatggac
caagcagtac tgtagatagt caaggttctg aaagtgaagc tctcccaatc
1920aactctaaaa agagttccgc actagaagaa acatattatc atctcctaaa
aacacagggt 1980ccatttgagg caatcaatta ttatcaccta atgagtgatg
aacccattgc ttttagcact 2040gaaagtggca aggaatatat ctttccagac
tcccttgaag aagcctaccc gccgtggttg 2100agtgagaagg aggccttaga
gaaggaaaat cgttatctgg tcattgatgg ccagcaattc 2160ctctggccgg
taatgagcct acgggacaag ttccttgctg ttcttcaaca tgactga
221710738PRTEbola virus 10Met Asp Lys Arg Val Arg Gly Ser Trp Ala
Leu Gly Gly Gln Ser Glu 1 5 10 15 Val Asp Leu Asp Tyr His Lys Ile
Leu Thr Ala Gly Leu Ser Val Gln 20 25 30 Gln Gly Ile Val Arg Gln
Arg Val Ile Pro Val Tyr Val Val Asn Asp 35 40 45 Leu Glu Gly Ile
Cys Gln His Ile Ile Gln Ala Phe Glu Ala Gly Val 50 55 60 Asp Phe
Gln Asp Asn Ala Asp Ser Phe Leu Leu Leu Leu Cys Leu His 65 70 75 80
His Ala Tyr Gln Gly Asp His Arg Leu Phe Leu Lys Ser Asp Ala Val 85
90 95 Gln Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Arg Glu Lys
Glu 100 105 110 Asn Val His Arg Leu Asp Glu Leu Leu Pro Asn Val Thr
Gly Gly Lys 115 120 125 Asn Leu Arg Arg Thr Leu Ala Ala Met Pro Glu
Glu Glu Thr Thr Glu 130 135 140 Ala Asn Ala Gly Gln Phe Leu Ser Phe
Ala Ser Leu Phe Leu Pro Lys 145 150 155 160 Leu Val Val Gly Glu Lys
Ala Cys Leu Glu Lys Val Gln Arg Gln Ile 165 170 175 Gln Val His Ala
Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ser Trp Gln 180 185 190 Ser Val
Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe 195 200 205
Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly 210
215 220 His Asp Ala Asn Asp Thr Val Ile Ser Asn Ser Val Ala Gln Ala
Arg 225 230 235 240 Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp
His Ile Leu Gln 245 250 255 Lys Thr Asp Leu Gly Val Arg Leu His Pro
Leu Ala Arg Thr Ala Lys 260 265 270 Val Lys Asn Glu Val Ser Ser Phe
Lys Ala Ala Leu Gly Ser Leu Ala 275 280 285 Lys His Gly Glu Tyr Ala
Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295 300 Val Asn Asn Leu
Glu His Gly Leu Tyr Pro Gln Leu Ser Ala Ile Ala 305 310 315 320 Leu
Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val 325 330
335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys
340 345 350 Gln Leu Gln Gln Tyr Ala Glu Thr Arg Glu Leu Asp Asn Leu
Gly Leu 355 360 365 Asp Glu Gln Glu Lys Lys Ile Leu Met Ser Phe His
Gln Lys Lys Asn 370 375 380 Glu Ile Ser Phe Gln Gln Thr Asn Ala Met
Val Thr Leu Arg Lys Glu 385 390 395 400 Arg Leu Ala Lys Leu Thr Glu
Ala Ile Thr Thr Ala Ser Lys Ile Lys 405 410 415 Val Gly Asp Arg Tyr
Pro Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro 420 425 430 Ile Tyr Asp
Asp Thr His Pro Asn Pro Ser Asp Asp Asn Pro Asp Asp 435 440 445 Ser
Arg Asp Thr Thr Ile Pro Gly Gly Val Val Asp Pro Tyr Asp Asp 450 455
460 Glu Ser Asn Asn Tyr Pro Asp Tyr Glu Asp Ser Ala Glu Gly Thr Thr
465 470 475 480 Gly Asp Leu Asp Leu Phe Asn Leu Asp Asp Asp Asp Asp
Asp Ser Arg 485 490 495 Pro Gly Pro Pro Asp Arg Gly Gln Asn Lys Glu
Arg Ala Ala Arg Thr 500 505 510 Tyr Gly Leu Gln Asp Pro Thr Leu Asp
Gly Ala Lys Lys Val Pro Glu 515 520 525 Leu Thr Pro Gly Ser His Gln
Pro Gly Asn Leu His Ile Thr Lys Ser 530 535 540 Gly Ser Asn Thr Asn
Gln Pro Gln Gly Asn Met Ser Ser Thr Leu His 545 550 555 560 Ser Met
Thr Pro Ile Gln Glu Glu Ser Glu Pro Asp Asp Gln Lys Asp 565 570 575
Asn Asp Asp Glu Ser Leu Thr Ser Leu Asp Ser Glu Gly Asp Glu Asp 580
585 590 Gly Glu Ser Ile Ser Glu Glu Asn Thr Pro Thr Val Ala Pro Pro
Ala 595 600 605 Pro Val Tyr Lys Asp Thr Gly Val Asp Thr Asn Gln Gln
Asn Gly Pro 610 615 620 Ser Ser Thr Val Asp Ser Gln Gly Ser Glu Ser
Glu Ala Leu Pro Ile 625 630 635 640 Asn Ser Lys Lys Ser Ser Ala Leu
Glu Glu Thr Tyr Tyr His Leu Leu 645 650 655 Lys Thr Gln Gly Pro Phe
Glu Ala Ile Asn Tyr Tyr His Leu Met Ser 660 665 670 Asp Glu Pro Ile
Ala Phe Ser Thr Glu Ser Gly Lys Glu Tyr Ile Phe 675 680 685 Pro Asp
Ser Leu Glu Glu Ala Tyr Pro Pro Trp Leu Ser Glu Lys Glu 690 695 700
Ala Leu Glu Lys Glu Asn Arg Tyr Leu Val Ile Asp Gly Gln Gln Phe 705
710 715 720 Leu Trp Pro Val Met Ser Leu Arg Asp Lys Phe Leu Ala Val
Leu Gln 725 730 735 His Asp 11465DNAEbola virus 11ctcaacgaga
cagaagacga tgatgcgaca tcgtcgagaa ctacaaaggg aagaatctcc 60gaccgggcca
ccaggaagta ttcggacctg gttccaaagg attcccctgg gatggtttca
120ttgcacgtac cagaagggga aacaacattg ccgtctcaga attcgacaga
aggtcgaaga 180gtagatgtga atactcagga aactatcaca gagacaactg
caacaatcat aggcactaac 240ggtaacaaca tgcagatctc caccatcggg
acaggactga gctccagcca aatcctgagt 300tcctcaccga ccatggcacc
aagccctgag actcagacct ccacaaccta cacaccaaaa 360ctaccagtga
tgaccaccga ggaatcaaca acaccaccga gaaactctcc tggctcaaca
420acagaagcac ccactctcac caccccagag aatataacat agtag
46512153PRTEbola virus 12Leu Asn Glu Thr Glu Asp Asp Asp Ala Thr
Ser Ser Arg Thr Thr Lys 1 5 10 15 Gly Arg Ile Ser Asp Arg Ala Thr
Arg Lys Tyr Ser Asp Leu Val Pro 20 25 30 Lys Asp Ser Pro Gly Met
Val Ser Leu His Val Pro Glu Gly Glu Thr 35 40 45 Thr Leu Pro Ser
Gln Asn Ser Thr Glu Gly Arg Arg Val Asp Val Asn 50 55 60 Thr Gln
Glu Thr Ile Thr Glu Thr Thr Ala Thr Ile Ile Gly Thr Asn 65 70 75 80
Gly Asn Asn Met Gln Ile Ser Thr Ile Gly Thr Gly Leu Ser Ser Ser 85
90 95 Gln Ile Leu Ser Ser Ser Pro Thr Met Ala Pro Ser Pro Glu Thr
Gln 100 105 110 Thr Ser Thr Thr Tyr Thr Pro Lys Leu Pro Val Met Thr
Thr Glu Glu 115 120 125 Ser Thr Thr Pro Pro Arg Asn Ser Pro Gly Ser
Thr Thr Glu Ala Pro 130 135 140 Thr Leu Thr Thr Pro Glu Asn Ile Thr
145 150 13981DNAEbola virus 13atgaggaggg caattctacc tactgcaccg
ccagaataca tagaggctgt ctacccaatg 60agaacggtta gtactagtat caacagtact
gccagtggtc cgaactttcc agcaccggat 120gtaatgatga gtgatacacc
ctccaactca ctccgaccaa ttgctgatga taacatcgat 180catccaagtc
atacaccaac cagtgtttca tcagccttta tactcgaggc aatggtgaat
240gtgatatcgg ggccgaaggt actaatgaag caaattccta tatggctccc
cttgggtgtt 300gctgatcaaa aaacatatag ttttgactca actacagctg
caattatgct cgcatcgtac 360accatcactc actttggcaa aacctccaat
ccgcttgtga gaatcaatcg acttggtcct 420gggatccccg atcacccgtt
gcggcttcta agaataggaa atcaagcctt cttgcaagag 480tttgtgctgc
ctccagttca attgccgcag tatttcactt ttgacctgac ggctctaaag
540ctgatcactc aacctctccc ggcagcaacc tggacggatg atactccgac
cggtcctaca 600ggaatacttc gtcctggaat ttcctttcat cccaaactga
gacctatcct attgccaggg 660aagaccggga aaagaggatc cagctccgat
cttacttctc ctgataaaat acaagcaata 720atgaactttc tccaagacct
caaactcgtg ccgattgatc cagccaagaa cattatgggt 780attgaagtgc
cggaactctt ggtccacaga ctaactggaa agaaaatcac aacaaaaaat
840ggtcaaccaa taattcctat tcttctacca aagtatattg gcatggatcc
catttctcag 900ggagacctca caatggtcat cactcaagac tgtgacactt
gccattctcc tgctagtctt 960cctccagtca gcgagaaatg a 98114326PRTEbola
virus 14Met Arg Arg Ala Ile Leu Pro Thr Ala Pro Pro Glu Tyr Ile Glu
Ala 1 5 10 15 Val Tyr Pro Met Arg Thr Val Ser Thr Ser Ile Asn Ser
Thr Ala Ser 20 25 30 Gly Pro Asn Phe Pro Ala Pro Asp Val Met Met
Ser Asp Thr Pro Ser 35 40 45 Asn Ser Leu Arg Pro Ile Ala Asp Asp
Asn Ile Asp His Pro Ser His 50 55 60 Thr Pro Thr Ser Val Ser Ser
Ala Phe Ile Leu Glu Ala Met Val Asn 65 70 75 80 Val Ile Ser Gly Pro
Lys Val Leu Met Lys Gln Ile Pro Ile Trp Leu 85 90 95 Pro Leu Gly
Val Ala Asp Gln Lys Thr Tyr Ser Phe Asp Ser Thr Thr 100 105 110 Ala
Ala Ile Met Leu Ala Ser Tyr Thr Ile Thr His Phe Gly Lys Thr 115 120
125 Ser Asn Pro Leu Val Arg Ile Asn Arg Leu Gly Pro Gly Ile Pro Asp
130 135 140 His Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu
Gln Glu 145 150 155 160 Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr
Phe Thr Phe Asp Leu 165 170 175 Thr Ala Leu Lys Leu Ile Thr Gln Pro
Leu Pro Ala Ala Thr Trp Thr 180 185 190 Asp Asp Thr Pro Thr Gly Pro
Thr Gly Ile Leu Arg Pro Gly Ile Ser 195 200 205 Phe His Pro Lys Leu
Arg Pro Ile Leu Leu Pro Gly Lys Thr Gly Lys 210 215 220 Arg Gly Ser
Ser Ser Asp Leu Thr Ser Pro Asp Lys Ile Gln Ala Ile 225 230 235 240
Met Asn Phe Leu Gln Asp Leu Lys Leu Val Pro Ile Asp Pro Ala Lys 245
250 255 Asn Ile Met Gly Ile Glu Val Pro Glu Leu Leu Val His Arg Leu
Thr 260 265 270 Gly Lys Lys Ile Thr Thr Lys Asn Gly Gln Pro Ile Ile
Pro Ile Leu 275 280 285 Leu Pro Lys Tyr Ile Gly Met Asp Pro Ile Ser
Gln Gly Asp Leu Thr 290 295 300 Met Val Ile Thr Gln Asp Cys Asp Thr
Cys His Ser Pro Ala Ser Leu 305 310 315 320 Pro Pro Val Ser Glu Lys
325 152220DNAEbola virus 15atggatcctc gtccaatcag aacctggatg
atgcataaca catctgaagt tgaagcagac 60taccataaga ttctaactgc cggattgtcc
gtccagcaag gcattgtgag acaaagaatc 120attcctgttt accaaatctc
aaacctggag gaagtatgtc aactcatcat acaggcattc 180gaggctggcg
tcgacttcca ggatagtgca gatagctttt tgttaatgct atgtctgcat
240catgcctatc aaggggatta taaacaattt ttggaaagta atgcggtaaa
ataccttgaa 300ggtcatggat tccgttttga gatgaagaaa aaggaaggtg
tcaagcgcct ggaggaacta 360ctccctgctg cctcgagtgg aaagaacatc
aagagaacat tggctgcaat gcccgaggag 420gaaacaacag aagcaaatgc
tggacaattt ctttcatttg ctagtctgtt tctcccaaaa 480ttggttgtcg
gagaaaaggc ctgtctggag aaggttcaac gacaaatcca agtgcacgca
540gaacaaggtc tgattcaata cccgacatct tggcaatcgg tgggacatat
gatggtcatc 600ttcagactaa tgcgaaccaa cttcctgatt aagttcctcc
taatacatca aggaatgcat 660atggttgcag ggcatgatgc taatgatgcc
gtcattgcca actctgtagc tcaagctcgt 720ttctccggat tgttgatagt
caaaacagtg cttgatcata tcctccaaaa aacagagcac 780ggagttcgcc
tgcatccctt ggcgcgaaca gccaaagtca aaaatgaggt gagctctttt
840aaggccgctt tagcctcact agcacaacat ggagaatatg ccccgtttgc
tcgtctgctg 900aatctatctg gggttaataa tcttgagcat gggcttttcc
ctcaactttc tgcaattgct 960ttgggagtag caactgcaca tgggagcact
ctggctggag tcaatgtagg agagcaatac 1020caacaactgc gagaagcagc
cactgaggcc gaaaagcagt tgcagaaata tgctgaatct 1080cgtgaacttg
atcacctagg tcttgatgat caggaaaaga aaatcctaaa agacttccat
1140cagaaaaaga atgagatcag cttccagcag acgacagcca tggtcacact
gcggaaagag 1200agattggcca aattgaccga agctattact tccacctcta
tcctcaaaac aggaaggcgg 1260tatgatgatg acaatgatat accctttcca
gggccaatca atgataacga gaactctggt 1320cagaacgatg acgatccaac
agactcccag gataccacaa tcccggatgt aataatcgat 1380ccaaacgatg
gtgggtataa taattacagc gattatgcaa atgatgctgc aagtgctcct
1440gatgacctag ttctttttga ccttgaggac gaggatgatg ctgataaccc
ggctcaaaac 1500acgccagaaa aaaatgatag accagcaaca acaaagctga
gaaatggaca ggaccaggat 1560ggaaaccaag gcgaaactgc atccccacgg
gtagccccca accaatacag agacaagcca 1620atgccacaag tacaggacag
atccgaaaat catgaccaaa cccttcaaac acagtccagg 1680gttttgactc
ctatcagcga ggaagcagac cccagcgacc acaacgatgg tgacaatgaa
1740agcattcctc ccctggaatc agacgacgag ggtagcactg atactactgc
agcagaaaca 1800aagcctgcca ctgcacctcc cgctcccgtc taccgaagta
tctccgtaga tgattctgtc 1860ccctcagaga acattcccgc acagtccaat
caaacgaaca atgaggacaa tgtcaggaac 1920aatgctcagt cggagcaatc
cattgcagaa atgtatcaac atatcttgaa aacacaagga 1980ccttttgatg
ccatccttta ctaccatatg atgaaagaag agcccatcat tttcagcact
2040agtgatggga aggagtatac atatccagac tctcttgaag atgagtatcc
accctggctc 2100agcgagaagg aagccatgaa cgaagacaat agattcataa
ccatggatgg tcagcagttt 2160tactggcctg tgatgaatca tagaaataaa
ttcatggcaa tcctccagca tcacaggtga 222016739PRTEbola virus 16Met Asp
Pro Arg Pro Ile Arg Thr Trp Met Met His Asn Thr Ser Glu 1 5 10 15
Val Glu Ala Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln 20
25 30 Gln Gly Ile Val Arg Gln Arg Ile Ile Pro Val Tyr Gln Ile Ser
Asn 35 40 45 Leu Glu Glu Val Cys Gln Leu Ile Ile Gln Ala Phe Glu
Ala Gly Val 50 55 60 Asp Phe Gln Asp Ser Ala Asp Ser Phe Leu Leu
Met Leu Cys Leu His 65 70 75 80 His Ala Tyr Gln Gly Asp Tyr Lys Gln
Phe Leu Glu Ser Asn Ala Val 85 90 95 Lys Tyr Leu Glu Gly His Gly
Phe Arg Phe Glu Met Lys Lys Lys Glu 100 105 110 Gly Val Lys Arg Leu
Glu Glu Leu Leu Pro Ala Ala Ser Ser Gly Lys 115 120 125 Asn Ile Lys
Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu 130 135 140 Ala
Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys 145 150
155 160 Leu Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln
Ile 165
170 175 Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ser Trp
Gln 180 185 190 Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg
Thr Asn Phe 195 200 205 Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met
His Met Val Ala Gly 210 215 220 His Asp Ala Asn Asp Ala Val Ile Ala
Asn Ser Val Ala Gln Ala Arg 225 230 235 240 Phe Ser Gly Leu Leu Ile
Val Lys Thr Val Leu Asp His Ile Leu Gln 245 250 255 Lys Thr Glu His
Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys 260 265 270 Val Lys
Asn Glu Val Ser Ser Phe Lys Ala Ala Leu Ala Ser Leu Ala 275 280 285
Gln His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290
295 300 Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile
Ala 305 310 315 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala
Gly Val Asn Val 325 330 335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala
Ala Thr Glu Ala Glu Lys 340 345 350 Gln Leu Gln Lys Tyr Ala Glu Ser
Arg Glu Leu Asp His Leu Gly Leu 355 360 365 Asp Asp Gln Glu Lys Lys
Ile Leu Lys Asp Phe His Gln Lys Lys Asn 370 375 380 Glu Ile Ser Phe
Gln Gln Thr Thr Ala Met Val Thr Leu Arg Lys Glu 385 390 395 400 Arg
Leu Ala Lys Leu Thr Glu Ala Ile Thr Ser Thr Ser Ile Leu Lys 405 410
415 Thr Gly Arg Arg Tyr Asp Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro
420 425 430 Ile Asn Asp Asn Glu Asn Ser Gly Gln Asn Asp Asp Asp Pro
Thr Asp 435 440 445 Ser Gln Asp Thr Thr Ile Pro Asp Val Ile Ile Asp
Pro Asn Asp Gly 450 455 460 Gly Tyr Asn Asn Tyr Ser Asp Tyr Ala Asn
Asp Ala Ala Ser Ala Pro 465 470 475 480 Asp Asp Leu Val Leu Phe Asp
Leu Glu Asp Glu Asp Asp Ala Asp Asn 485 490 495 Pro Ala Gln Asn Thr
Pro Glu Lys Asn Asp Arg Pro Ala Thr Thr Lys 500 505 510 Leu Arg Asn
Gly Gln Asp Gln Asp Gly Asn Gln Gly Glu Thr Ala Ser 515 520 525 Pro
Arg Val Ala Pro Asn Gln Tyr Arg Asp Lys Pro Met Pro Gln Val 530 535
540 Gln Asp Arg Ser Glu Asn His Asp Gln Thr Leu Gln Thr Gln Ser Arg
545 550 555 560 Val Leu Thr Pro Ile Ser Glu Glu Ala Asp Pro Ser Asp
His Asn Asp 565 570 575 Gly Asp Asn Glu Ser Ile Pro Pro Leu Glu Ser
Asp Asp Glu Gly Ser 580 585 590 Thr Asp Thr Thr Ala Ala Glu Thr Lys
Pro Ala Thr Ala Pro Pro Ala 595 600 605 Pro Val Tyr Arg Ser Ile Ser
Val Asp Asp Ser Val Pro Ser Glu Asn 610 615 620 Ile Pro Ala Gln Ser
Asn Gln Thr Asn Asn Glu Asp Asn Val Arg Asn 625 630 635 640 Asn Ala
Gln Ser Glu Gln Ser Ile Ala Glu Met Tyr Gln His Ile Leu 645 650 655
Lys Thr Gln Gly Pro Phe Asp Ala Ile Leu Tyr Tyr His Met Met Lys 660
665 670 Glu Glu Pro Ile Ile Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr
Tyr 675 680 685 Pro Asp Ser Leu Glu Asp Glu Tyr Pro Pro Trp Leu Ser
Glu Lys Glu 690 695 700 Ala Met Asn Glu Asp Asn Arg Phe Ile Thr Met
Asp Gly Gln Gln Phe 705 710 715 720 Tyr Trp Pro Val Met Asn His Arg
Asn Lys Phe Met Ala Ile Leu Gln 725 730 735 His His Arg
17465DNAEbola virus 17agagcccagg atccaggtag caaccagaag acgaaggtca
ctcccaccag cttcgccaac 60aaccaaacct ccaagaacca cgaagacttg gttccagagg
atcccgcttc agtggttcaa 120gtgcgagacc tccagaggga aaacacagtg
ccgaccccac ccccagacac agtccccaca 180actctgatcc ccgacacaat
ggaggaacaa accaccagcc actacgaacc accaaacatt 240tccagaaacc
atcaagagag gaacaacacc gcacaccccg aaactctcgc caacaatccc
300ccagacaaca caaccccgtc gacaccacct caagacggtg agcggacaag
ttcccacaca 360acaccctccc cccgcccagt cccaaccagc acaatccatc
ccaccacgcg agagactcac 420attcccacca caatgacaac aagccatgac
accgacagct agtag 46518153PRTEbola virus 18Arg Ala Gln Asp Pro Gly
Ser Asn Gln Lys Thr Lys Val Thr Pro Thr 1 5 10 15 Ser Phe Ala Asn
Asn Gln Thr Ser Lys Asn His Glu Asp Leu Val Pro 20 25 30 Glu Asp
Pro Ala Ser Val Val Gln Val Arg Asp Leu Gln Arg Glu Asn 35 40 45
Thr Val Pro Thr Pro Pro Pro Asp Thr Val Pro Thr Thr Leu Ile Pro 50
55 60 Asp Thr Met Glu Glu Gln Thr Thr Ser His Tyr Glu Pro Pro Asn
Ile 65 70 75 80 Ser Arg Asn His Gln Glu Arg Asn Asn Thr Ala His Pro
Glu Thr Leu 85 90 95 Ala Asn Asn Pro Pro Asp Asn Thr Thr Pro Ser
Thr Pro Pro Gln Asp 100 105 110 Gly Glu Arg Thr Ser Ser His Thr Thr
Pro Ser Pro Arg Pro Val Pro 115 120 125 Thr Ser Thr Ile His Pro Thr
Thr Arg Glu Thr His Ile Pro Thr Thr 130 135 140 Met Thr Thr Ser His
Asp Thr Asp Ser 145 150 19981DNAEbola virus 19atgaggagaa tcatcctacc
cacggcacca cctgaataca tggaggctgt ttacccaatg 60agaacaatga attctggtgc
agacaacact gccagtggcc ctaattacac aacaactggt 120gtgatgacaa
atgatactcc ctctaattca ctccgaccag ttgcagatga taatattgat
180catccgagcc acacgcctaa cagtgttgcc tctgcattta tattggaagc
tatggtgaat 240gtaatatctg gcccgaaagt gctgatgaag caaatcccaa
tctggcttcc tctgggtgtc 300tctgaccaga agacatatag ctttgattca
accactgctg ccattatgct agcatcatat 360accatcactc attttggcaa
aacctcaaat ccccttgtga gaatcaaccg acttggtcct 420ggcatacctg
atcacccact acgactccta agaataggaa atcaagcctt cctacaagag
480tttgtgctac ctcctgtaca actgccacaa tacttcactt ttgatctgac
agcgctgaag 540ctgatcaccc agccactccc agcggcaacc tggacagatg
aaactccagc tgtgtcaact 600ggcacgctcc gcccagggat ctcattccat
cccaaattaa ggcctatcct gctaccagga 660agagctggaa agaagggctc
caactccgat ctaacatctc ctgacaaaat ccaggctata 720atgaatttcc
tacaagacct caaaattgta ccaatcgatc caaccaagaa tatcatgggt
780attgaagtgc cagaactcct ggttcacagg ctgactggga agaagacaac
taccaagaat 840ggtcaaccaa tcattccaat tctgctacca aagtacattg
gtcttgatcc tctatctcaa 900ggtgatctca caatggtgat cactcaggac
tgtgattcct gccactcccc ggccagtctt 960cccccagtca atgaaaaatg a
98120326PRTEbola virus 20Met Arg Arg Ile Ile Leu Pro Thr Ala Pro
Pro Glu Tyr Met Glu Ala 1 5 10 15 Val Tyr Pro Met Arg Thr Met Asn
Ser Gly Ala Asp Asn Thr Ala Ser 20 25 30 Gly Pro Asn Tyr Thr Thr
Thr Gly Val Met Thr Asn Asp Thr Pro Ser 35 40 45 Asn Ser Leu Arg
Pro Val Ala Asp Asp Asn Ile Asp His Pro Ser His 50 55 60 Thr Pro
Asn Ser Val Ala Ser Ala Phe Ile Leu Glu Ala Met Val Asn 65 70 75 80
Val Ile Ser Gly Pro Lys Val Leu Met Lys Gln Ile Pro Ile Trp Leu 85
90 95 Pro Leu Gly Val Ser Asp Gln Lys Thr Tyr Ser Phe Asp Ser Thr
Thr 100 105 110 Ala Ala Ile Met Leu Ala Ser Tyr Thr Ile Thr His Phe
Gly Lys Thr 115 120 125 Ser Asn Pro Leu Val Arg Ile Asn Arg Leu Gly
Pro Gly Ile Pro Asp 130 135 140 His Pro Leu Arg Leu Leu Arg Ile Gly
Asn Gln Ala Phe Leu Gln Glu 145 150 155 160 Phe Val Leu Pro Pro Val
Gln Leu Pro Gln Tyr Phe Thr Phe Asp Leu 165 170 175 Thr Ala Leu Lys
Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr Trp Thr 180 185 190 Asp Glu
Thr Pro Ala Val Ser Thr Gly Thr Leu Arg Pro Gly Ile Ser 195 200 205
Phe His Pro Lys Leu Arg Pro Ile Leu Leu Pro Gly Arg Ala Gly Lys 210
215 220 Lys Gly Ser Asn Ser Asp Leu Thr Ser Pro Asp Lys Ile Gln Ala
Ile 225 230 235 240 Met Asn Phe Leu Gln Asp Leu Lys Ile Val Pro Ile
Asp Pro Thr Lys 245 250 255 Asn Ile Met Gly Ile Glu Val Pro Glu Leu
Leu Val His Arg Leu Thr 260 265 270 Gly Lys Lys Thr Thr Thr Lys Asn
Gly Gln Pro Ile Ile Pro Ile Leu 275 280 285 Leu Pro Lys Tyr Ile Gly
Leu Asp Pro Leu Ser Gln Gly Asp Leu Thr 290 295 300 Met Val Ile Thr
Gln Asp Cys Asp Ser Cys His Ser Pro Ala Ser Leu 305 310 315 320 Pro
Pro Val Asn Glu Lys 325 212220DNAEbola virus 21atggagagtc
gggcccacaa agcatggatg acgcacaccg catcaggttt cgaaacagat 60taccataaga
ttttaacagc aggattgtca gtccaacaag gcattgtgag acaacgggtc
120attcaagtcc accaggttac aaacctagaa gaaatatgcc aattgatcat
tcaagccttt 180gaagctggtg ttgattttca agagagtgca gacagtttct
tgctgatgct atgtttacat 240catgcttatc agggtgacta caagcaattc
ttggaaagca atgcagtcaa gtaccttgag 300ggtcatggct ttcgctttga
ggtcaggaaa aaggaaggag tcaagcgact cgaagaattg 360cttcctgctg
catccagtgg caagagcatc aggagaacac tggctgcaat gcctgaagag
420gagacaacag aagcaaatgc cggacagttc ctctcttttg ctagcttatt
tcttcctaag 480ctagttgtcg gagaaaaagc ctgtctagaa aaggtgcagc
ggcaaattca agttcattct 540gagcagggat tgatccaata ccccacagcc
tggcagtcag ttggacacat gatggtcatt 600ttcagactga tgagaacaaa
ttttctaatt aagttcctcc ttatacatca agggatgcat 660atggtagcag
gacacgatgc taacgatgct gtcatcgcaa actctgtagc tcaagcacgt
720ttttcaggat tattgatcgt taaaacagtg ctagatcaca tccttcagaa
aacagagcac 780ggagtgcgtc ttcatccttt ggcaagaact gctaaggtca
agaacgaagt aaattccttt 840aaggctgccc ttagctcgct agcacaacat
ggagagtatg ctccttttgc tcgcttgctg 900aatctttctg gagtcaacaa
tctcgagcac ggactgtttc ctcagctttc tgcaattgcc 960ctaggtgtcg
caacggcaca cggcagtacc ctggcaggag taaatgtggg ggaacagtat
1020cagcaactac gagaagcagc cactgaggca gaaaaacaat tgcagaaata
cgctgaatct 1080cgcgagcttg accatctagg tctcgatgat caagagaaga
agatcttgaa agacttccat 1140cagaagaaaa atgaaatcag cttccagcag
acaacagcca tggtcacact acggaaggaa 1200aggctagcca agctcactga
ggcaatcacc tccacatccc ttctcaagac aggaaaacag 1260tatgatgatg
acaacgatat cccctttcct gggcccatca atgataacga aaactcagaa
1320cagcaagacg atgatccaac agattctcag gacactacca tccctgatat
cattgttgac 1380ccggatgatg gcagatacaa caattatgga gactatccta
gtgagacggc gaatgcccct 1440gaagaccttg ttctttttga ccttgaagat
ggtgacgagg atgatcaccg accgtcaagt 1500tcatcagaga acaacaacaa
acacagtctt acaggaactg acagtaacaa aacaagtaac 1560tggaatcgaa
acccgactaa tatgccaaag aaagactcca cacaaaacaa tgacaatcct
1620gcacagcggg ctcaagaata cgccagggat aacatccagg atacaccaac
accccatcga 1680gctctaactc ccatcagcga agaaaccggc tccaatggtc
acaatgaaga tgacattgat 1740agcatccctc ctttggaatc agacgaagaa
aacaacactg agacaaccat taccaccaca 1800aaaaatacca ctgctccacc
agcacctgtt tatcggagta attcagaaaa ggagcccctc 1860ccgcaagaaa
aatcccagaa gcaaccaaac caagtgagtg gtagtgagaa taccgacaat
1920aaacctcact cagagcaatc agtggaagaa atgtatcgac acatcctcca
aacacaagga 1980ccatttgatg ccatcctata ctattacatg atgacggagg
agccgattgt ctttagcact 2040agtgatggga aagaatacgt ataccctgat
tctcttgaag gggagcatcc accgtggctc 2100agtgaaaaag aggccttgaa
tgaggacaat aggtttatca caatggatga tcaacaattc 2160tactggcctg
taatgaatca caggaacaaa ttcatggcta tccttcagca ccacaagtaa
222022739PRTEbola virus 22Met Glu Ser Arg Ala His Lys Ala Trp Met
Thr His Thr Ala Ser Gly 1 5 10 15 Phe Glu Thr Asp Tyr His Lys Ile
Leu Thr Ala Gly Leu Ser Val Gln 20 25 30 Gln Gly Ile Val Arg Gln
Arg Val Ile Gln Val His Gln Val Thr Asn 35 40 45 Leu Glu Glu Ile
Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val 50 55 60 Asp Phe
Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His 65 70 75 80
His Ala Tyr Gln Gly Asp Tyr Lys Gln Phe Leu Glu Ser Asn Ala Val 85
90 95 Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Arg Lys Lys
Glu 100 105 110 Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Ala Ser
Ser Gly Lys 115 120 125 Ser Ile Arg Arg Thr Leu Ala Ala Met Pro Glu
Glu Glu Thr Thr Glu 130 135 140 Ala Asn Ala Gly Gln Phe Leu Ser Phe
Ala Ser Leu Phe Leu Pro Lys 145 150 155 160 Leu Val Val Gly Glu Lys
Ala Cys Leu Glu Lys Val Gln Arg Gln Ile 165 170 175 Gln Val His Ser
Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln 180 185 190 Ser Val
Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe 195 200 205
Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly 210
215 220 His Asp Ala Asn Asp Ala Val Ile Ala Asn Ser Val Ala Gln Ala
Arg 225 230 235 240 Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp
His Ile Leu Gln 245 250 255 Lys Thr Glu His Gly Val Arg Leu His Pro
Leu Ala Arg Thr Ala Lys 260 265 270 Val Lys Asn Glu Val Asn Ser Phe
Lys Ala Ala Leu Ser Ser Leu Ala 275 280 285 Gln His Gly Glu Tyr Ala
Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295 300 Val Asn Asn Leu
Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala 305 310 315 320 Leu
Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val 325 330
335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys
340 345 350 Gln Leu Gln Lys Tyr Ala Glu Ser Arg Glu Leu Asp His Leu
Gly Leu 355 360 365 Asp Asp Gln Glu Lys Lys Ile Leu Lys Asp Phe His
Gln Lys Lys Asn 370 375 380 Glu Ile Ser Phe Gln Gln Thr Thr Ala Met
Val Thr Leu Arg Lys Glu 385 390 395 400 Arg Leu Ala Lys Leu Thr Glu
Ala Ile Thr Ser Thr Ser Leu Leu Lys 405 410 415 Thr Gly Lys Gln Tyr
Asp Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro 420 425 430 Ile Asn Asp
Asn Glu Asn Ser Glu Gln Gln Asp Asp Asp Pro Thr Asp 435 440 445 Ser
Gln Asp Thr Thr Ile Pro Asp Ile Ile Val Asp Pro Asp Asp Gly 450 455
460 Arg Tyr Asn Asn Tyr Gly Asp Tyr Pro Ser Glu Thr Ala Asn Ala Pro
465 470 475 480 Glu Asp Leu Val Leu Phe Asp Leu Glu Asp Gly Asp Glu
Asp Asp His 485 490 495 Arg Pro Ser Ser Ser Ser Glu Asn Asn Asn Lys
His Ser Leu Thr Gly 500 505 510 Thr Asp Ser Asn Lys Thr Ser Asn Trp
Asn Arg Asn Pro Thr Asn Met 515 520 525 Pro Lys Lys Asp Ser Thr Gln
Asn Asn Asp Asn Pro Ala Gln Arg Ala 530 535 540 Gln Glu Tyr Ala Arg
Asp Asn Ile Gln Asp Thr Pro Thr Pro His Arg 545 550 555 560 Ala Leu
Thr Pro Ile Ser Glu Glu Thr Gly Ser Asn Gly His Asn Glu 565 570 575
Asp Asp Ile Asp Ser Ile Pro Pro Leu Glu Ser Asp Glu Glu Asn Asn 580
585 590 Thr Glu Thr Thr Ile Thr Thr Thr Lys Asn Thr Thr Ala Pro Pro
Ala 595 600 605 Pro Val Tyr Arg Ser Asn Ser Glu Lys Glu Pro Leu Pro
Gln Glu Lys 610 615 620 Ser Gln Lys Gln Pro Asn Gln Val Ser Gly Ser
Glu Asn Thr Asp Asn 625 630 635 640 Lys Pro His Ser Glu Gln Ser Val
Glu Glu Met Tyr Arg His Ile Leu 645 650 655 Gln Thr Gln Gly Pro Phe
Asp Ala Ile Leu Tyr Tyr Tyr Met Met Thr 660 665 670 Glu Glu Pro Ile
Val Phe Ser Thr Ser Asp Gly Lys Glu
Tyr Val Tyr 675 680 685 Pro Asp Ser Leu Glu Gly Glu His Pro Pro Trp
Leu Ser Glu Lys Glu 690 695 700 Ala Leu Asn Glu Asp Asn Arg Phe Ile
Thr Met Asp Asp Gln Gln Phe 705 710 715 720 Tyr Trp Pro Val Met Asn
His Arg Asn Lys Phe Met Ala Ile Leu Gln 725 730 735 His His Lys
23465DNAEbola virus 23gaaacccaga accaggtcct tgacacgaca gcgacggtct
ctcctcccat ctccgcccac 60aaccacgcag ccgaagacca caaagaattg gtttcagagg
attccactcc agtggttcag 120atgcaaaaca tcaagggaaa ggacacaatg
ccaaccacag tgacgggtgt accaacaacc 180acaccctctc catttccaat
caatgctcgc aacactgatc ataccaaatc atttatcggc 240ctggaggggc
cccaagaaga ccacagcacc acacagcctg ccaagaccac cagccaacca
300accaacagca cagaatcgac gacactaaac ccaacatcag agccctccag
tagaggcacg 360ggaccatcca gccccacggt ccccaacacc acagaaagcc
acgccgaact tggcaagaca 420accccaacca cactcccaga acagcacact
gccgccagtt agtag 46524153PRTEbola virus 24Glu Thr Gln Asn Gln Val
Leu Asp Thr Thr Ala Thr Val Ser Pro Pro 1 5 10 15 Ile Ser Ala His
Asn His Ala Ala Glu Asp His Lys Glu Leu Val Ser 20 25 30 Glu Asp
Ser Thr Pro Val Val Gln Met Gln Asn Ile Lys Gly Lys Asp 35 40 45
Thr Met Pro Thr Thr Val Thr Gly Val Pro Thr Thr Thr Pro Ser Pro 50
55 60 Phe Pro Ile Asn Ala Arg Asn Thr Asp His Thr Lys Ser Phe Ile
Gly 65 70 75 80 Leu Glu Gly Pro Gln Glu Asp His Ser Thr Thr Gln Pro
Ala Lys Thr 85 90 95 Thr Ser Gln Pro Thr Asn Ser Thr Glu Ser Thr
Thr Leu Asn Pro Thr 100 105 110 Ser Glu Pro Ser Ser Arg Gly Thr Gly
Pro Ser Ser Pro Thr Val Pro 115 120 125 Asn Thr Thr Glu Ser His Ala
Glu Leu Gly Lys Thr Thr Pro Thr Thr 130 135 140 Leu Pro Glu Gln His
Thr Ala Ala Ser 145 150 25996DNAEbola virus 25atgagacgcg gagtgttacc
aacggctcct ccagcatata atgatattgc ataccctatg 60agcatactcc caacccgacc
aagtgtcata gtcaatgaga ccaaatcaga tgtactggca 120gtgccagggg
cagatgttcc atcaaactcc atgagaccag tggctgatga taacattgat
180cactcaagcc atactccaag cggagtagct tctgccttta tattggaagc
tacagtgaat 240gtaatttcgg gaacaaaagt cctgatgaag caaataccta
tttggcttcc actgggtgta 300gctgatcaga agatatacag ctttgattca
acaacagccg caattatgtt ggcttcctac 360acagtgacac acttcgggaa
gatatctaac ccgctggtac gtgtcaacag gctaggccca 420ggaatacccg
atcatccgct acgactccta aggttgggca atcaggcatt ccttcaagag
480tttgttcttc caccagtcca gcttccccag tatttcacat ttgatctaac
agctctaaag 540ctcatcactc aaccattgcc agctgcaacc tggacagacg
aaactccagc aggagcagtc 600aatgctcttc gtcctgggct ctcactccat
cccaagcttc gtccaattct cctgccgggg 660aagacaggaa agaaaggaca
tgcttcagac ttaacatcac ctgacaagat tcaaacaatc 720atgaatgcaa
taccggacct caaaattgtc ccgattgatc caaccaagaa catagttgga
780attgaggttc cagaattact agttcaaagg ctgaccggca aaaaaccaca
acccaaaaat 840ggccaaccaa ttattccagt tcttcttccg aaatatgttg
gacttgatcc tatatcgcca 900ggggacttaa ctatggttat cacccaggat
tgtgattcat gccactctcc agccagccat 960ccgtatcaca tggacaagca
ggatagttac caataa 99626331PRTEbola virus 26Met Arg Arg Gly Val Leu
Pro Thr Ala Pro Pro Ala Tyr Asn Asp Ile 1 5 10 15 Ala Tyr Pro Met
Ser Ile Leu Pro Thr Arg Pro Ser Val Ile Val Asn 20 25 30 Glu Thr
Lys Ser Asp Val Leu Ala Val Pro Gly Ala Asp Val Pro Ser 35 40 45
Asn Ser Met Arg Pro Val Ala Asp Asp Asn Ile Asp His Ser Ser His 50
55 60 Thr Pro Ser Gly Val Ala Ser Ala Phe Ile Leu Glu Ala Thr Val
Asn 65 70 75 80 Val Ile Ser Gly Thr Lys Val Leu Met Lys Gln Ile Pro
Ile Trp Leu 85 90 95 Pro Leu Gly Val Ala Asp Gln Lys Ile Tyr Ser
Phe Asp Ser Thr Thr 100 105 110 Ala Ala Ile Met Leu Ala Ser Tyr Thr
Val Thr His Phe Gly Lys Ile 115 120 125 Ser Asn Pro Leu Val Arg Val
Asn Arg Leu Gly Pro Gly Ile Pro Asp 130 135 140 His Pro Leu Arg Leu
Leu Arg Leu Gly Asn Gln Ala Phe Leu Gln Glu 145 150 155 160 Phe Val
Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe Asp Leu 165 170 175
Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr Trp Thr 180
185 190 Asp Glu Thr Pro Ala Gly Ala Val Asn Ala Leu Arg Pro Gly Leu
Ser 195 200 205 Leu His Pro Lys Leu Arg Pro Ile Leu Leu Pro Gly Lys
Thr Gly Lys 210 215 220 Lys Gly His Ala Ser Asp Leu Thr Ser Pro Asp
Lys Ile Gln Thr Ile 225 230 235 240 Met Asn Ala Ile Pro Asp Leu Lys
Ile Val Pro Ile Asp Pro Thr Lys 245 250 255 Asn Ile Val Gly Ile Glu
Val Pro Glu Leu Leu Val Gln Arg Leu Thr 260 265 270 Gly Lys Lys Pro
Gln Pro Lys Asn Gly Gln Pro Ile Ile Pro Val Leu 275 280 285 Leu Pro
Lys Tyr Val Gly Leu Asp Pro Ile Ser Pro Gly Asp Leu Thr 290 295 300
Met Val Ile Thr Gln Asp Cys Asp Ser Cys His Ser Pro Ala Ser His 305
310 315 320 Pro Tyr His Met Asp Lys Gln Asp Ser Tyr Gln 325 330
272220DNAEbola virus 27atggatcgtg ggaccagaag aatctgggtg tcgcaaaatc
aaggtgatac tgatttagat 60tatcataaaa ttttgacagc tggccttact gttcaacagg
gaattgtcag gcagaaaata 120atttctgtat atcttgttga taacttggag
gctatgtgtc aattggtaat acaagccttt 180gaggccggaa ttgatttcca
agaaaatgcc gacagcttcc ttctgatgct ttgcctacat 240catgcttacc
aaggtgacta taaattgttc ttggagagca atgctgtaca gtatttggaa
300ggtcatggat tcaaatttga gctccggaag aaggacggtg tcaatcggct
cgaggaattg 360cttcctgctg caacgagtgg aaaaaacatc aggcgtacgt
tggccgcact gcctgaagag 420gagactacag aagcaaatgc agggcaattt
ctctcatttg cgagtttgtt tcttcccaaa 480ctggttgtgg gagagaaggc
ttgcttggaa aaagtccagc gacaaattca ggttcatgca 540gaacagggtt
taattcaata tcccactgca tggcaatcag ttggacacat gatggtaatc
600ttcagattga tgaggactaa tttcttgatt aaatatttac tgatccacca
gggtatgcat 660atggtagctg gccacgatgc caatgatgct gtcattgcta
attcagttgc tcaggctcgc 720ttttcaggac tcctaattgt caaaaccgtt
cttgatcata ttctgcaaaa aaccgaccaa 780ggagtaagac ttcacccttt
ggcccgaaca gccaaagtgc gtaatgaggt taatgcattt 840aaggccgccc
taagctcact tgctaagcat ggggaatatg ccccttttgc tcgccttctc
900aatctctcgg gagttaacaa cctagaacat ggtctctacc cacagttatc
agcaattgct 960cttggagttg ccacagcaca tggtagcacc cttgcaggag
ttaatgttgg tgagcagtat 1020cagcagctta gagaggctgc cactgaagct
gagaagcaac tccaacaata tgctgagtcc 1080agagaactcg acagcctagg
cctggacgat caggaaagaa gaatactaat gaacttccat 1140cagaagaaaa
acgaaattag tttccagcag accaatgcaa tggtaaccct taggaaagag
1200cgactggcta aattaacaga agctataacg ctggcctcaa gacctaacct
cgggtctaga 1260caagacgacg gcaatgaaat accgttccct gggcctataa
gcaacaaccc agaccaagat 1320catctggagg atgatcctag agactccaga
gacaccatca ttcctaatgg tgcaattgac 1380cccgaggatg gtgattttga
aaattacaat ggctatcatg atgatgaagt tgggacggca 1440ggtgacttgg
tcctgttcga tcttgacgat catgaggatg acaataaagc ttttgagcca
1500caggacagct cgccacaatc ccaaagggaa atagagagag aaagattaat
tcatccaccc 1560ccaggcaaca acaaggacga caatcgagcc tcagacaaca
atcaacaatc agcagattct 1620gaggaacaag gaggtcaata caactggcac
cgaggcccag aacgtacgac cgccaatcga 1680agactctcac cagtgcacga
agaggacacc cttatggatc aaggcgatga tgatccctca 1740agcttacctc
cgctggaatc tgatgatgac gatgcatcaa gtagccaaca agatcccgat
1800tatacagctg ttgcccctcc tgctcctgta taccgcagtg cagaagccca
cgagcctccc 1860cacaaatcct cgaacgagcc agctgaaaca tcacaattga
atgaagaccc tgatatcggt 1920caatcaaagt ctatgcaaaa attagaagag
acatatcacc atctgctgag aactcaaggt 1980ccatttgaag ccatcaatta
ttatcacatg atgaaggatg agccggtaat atttagcact 2040gatgatggga
aggaatacac ctacccggat tcacttgagg aagcctatcc tccatggctc
2100accgagaaag aacgactgga caaagagaat cgctacattt acataaataa
tcaacagttc 2160ttctggcctg tcatgagtcc cagagacaaa tttcttgcaa
tcttgcagca ccatcagtaa 222028739PRTEbola virus 28Met Asp Arg Gly Thr
Arg Arg Ile Trp Val Ser Gln Asn Gln Gly Asp 1 5 10 15 Thr Asp Leu
Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Thr Val Gln 20 25 30 Gln
Gly Ile Val Arg Gln Lys Ile Ile Ser Val Tyr Leu Val Asp Asn 35 40
45 Leu Glu Ala Met Cys Gln Leu Val Ile Gln Ala Phe Glu Ala Gly Ile
50 55 60 Asp Phe Gln Glu Asn Ala Asp Ser Phe Leu Leu Met Leu Cys
Leu His 65 70 75 80 His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu
Ser Asn Ala Val 85 90 95 Gln Tyr Leu Glu Gly His Gly Phe Lys Phe
Glu Leu Arg Lys Lys Asp 100 105 110 Gly Val Asn Arg Leu Glu Glu Leu
Leu Pro Ala Ala Thr Ser Gly Lys 115 120 125 Asn Ile Arg Arg Thr Leu
Ala Ala Leu Pro Glu Glu Glu Thr Thr Glu 130 135 140 Ala Asn Ala Gly
Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys 145 150 155 160 Leu
Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile 165 170
175 Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln
180 185 190 Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr
Asn Phe 195 200 205 Leu Ile Lys Tyr Leu Leu Ile His Gln Gly Met His
Met Val Ala Gly 210 215 220 His Asp Ala Asn Asp Ala Val Ile Ala Asn
Ser Val Ala Gln Ala Arg 225 230 235 240 Phe Ser Gly Leu Leu Ile Val
Lys Thr Val Leu Asp His Ile Leu Gln 245 250 255 Lys Thr Asp Gln Gly
Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys 260 265 270 Val Arg Asn
Glu Val Asn Ala Phe Lys Ala Ala Leu Ser Ser Leu Ala 275 280 285 Lys
His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295
300 Val Asn Asn Leu Glu His Gly Leu Tyr Pro Gln Leu Ser Ala Ile Ala
305 310 315 320 Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly
Val Asn Val 325 330 335 Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala
Thr Glu Ala Glu Lys 340 345 350 Gln Leu Gln Gln Tyr Ala Glu Ser Arg
Glu Leu Asp Ser Leu Gly Leu 355 360 365 Asp Asp Gln Glu Arg Arg Ile
Leu Met Asn Phe His Gln Lys Lys Asn 370 375 380 Glu Ile Ser Phe Gln
Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu 385 390 395 400 Arg Leu
Ala Lys Leu Thr Glu Ala Ile Thr Leu Ala Ser Arg Pro Asn 405 410 415
Leu Gly Ser Arg Gln Asp Asp Gly Asn Glu Ile Pro Phe Pro Gly Pro 420
425 430 Ile Ser Asn Asn Pro Asp Gln Asp His Leu Glu Asp Asp Pro Arg
Asp 435 440 445 Ser Arg Asp Thr Ile Ile Pro Asn Gly Ala Ile Asp Pro
Glu Asp Gly 450 455 460 Asp Phe Glu Asn Tyr Asn Gly Tyr His Asp Asp
Glu Val Gly Thr Ala 465 470 475 480 Gly Asp Leu Val Leu Phe Asp Leu
Asp Asp His Glu Asp Asp Asn Lys 485 490 495 Ala Phe Glu Pro Gln Asp
Ser Ser Pro Gln Ser Gln Arg Glu Ile Glu 500 505 510 Arg Glu Arg Leu
Ile His Pro Pro Pro Gly Asn Asn Lys Asp Asp Asn 515 520 525 Arg Ala
Ser Asp Asn Asn Gln Gln Ser Ala Asp Ser Glu Glu Gln Gly 530 535 540
Gly Gln Tyr Asn Trp His Arg Gly Pro Glu Arg Thr Thr Ala Asn Arg 545
550 555 560 Arg Leu Ser Pro Val His Glu Glu Asp Thr Leu Met Asp Gln
Gly Asp 565 570 575 Asp Asp Pro Ser Ser Leu Pro Pro Leu Glu Ser Asp
Asp Asp Asp Ala 580 585 590 Ser Ser Ser Gln Gln Asp Pro Asp Tyr Thr
Ala Val Ala Pro Pro Ala 595 600 605 Pro Val Tyr Arg Ser Ala Glu Ala
His Glu Pro Pro His Lys Ser Ser 610 615 620 Asn Glu Pro Ala Glu Thr
Ser Gln Leu Asn Glu Asp Pro Asp Ile Gly 625 630 635 640 Gln Ser Lys
Ser Met Gln Lys Leu Glu Glu Thr Tyr His His Leu Leu 645 650 655 Arg
Thr Gln Gly Pro Phe Glu Ala Ile Asn Tyr Tyr His Met Met Lys 660 665
670 Asp Glu Pro Val Ile Phe Ser Thr Asp Asp Gly Lys Glu Tyr Thr Tyr
675 680 685 Pro Asp Ser Leu Glu Glu Ala Tyr Pro Pro Trp Leu Thr Glu
Lys Glu 690 695 700 Arg Leu Asp Lys Glu Asn Arg Tyr Ile Tyr Ile Asn
Asn Gln Gln Phe 705 710 715 720 Phe Trp Pro Val Met Ser Pro Arg Asp
Lys Phe Leu Ala Ile Leu Gln 725 730 735 His His Gln 29465DNAEbola
virus 29acccacacca acaactcctc agatcagagc ccggcgggaa ctgtccaagg
aaaaattagc 60taccacccac ccgccaacaa ctccgagctg gttccaacgg attcccctcc
agtagtttca 120gtgctcactg caggacggac agaggaaatg tcgacccaag
gtctaaccaa cggagagaca 180atcacaggtt tcaccgcgaa cccaatgaca
accaccattg ccccaagtcc aaccatgaca 240agcgaggttg ataacaatgt
accaagtgaa caaccgaaca acacagcatc cattgaagac 300tcccccccat
cggcaagcaa cgagacaatt taccactccg agatggatcc gatccaaggc
360tcgaacaact ccgcccagag cccacagacc aagaccacgc cagcacccac
aacatccccg 420atgacccagg acccgcaaga gacggccaac agcagcaaat agtag
46530153PRTEbola virus 30Thr His Thr Asn Asn Ser Ser Asp Gln Ser
Pro Ala Gly Thr Val Gln 1 5 10 15 Gly Lys Ile Ser Tyr His Pro Pro
Ala Asn Asn Ser Glu Leu Val Pro 20 25 30 Thr Asp Ser Pro Pro Val
Val Ser Val Leu Thr Ala Gly Arg Thr Glu 35 40 45 Glu Met Ser Thr
Gln Gly Leu Thr Asn Gly Glu Thr Ile Thr Gly Phe 50 55 60 Thr Ala
Asn Pro Met Thr Thr Thr Ile Ala Pro Ser Pro Thr Met Thr 65 70 75 80
Ser Glu Val Asp Asn Asn Val Pro Ser Glu Gln Pro Asn Asn Thr Ala 85
90 95 Ser Ile Glu Asp Ser Pro Pro Ser Ala Ser Asn Glu Thr Ile Tyr
His 100 105 110 Ser Glu Met Asp Pro Ile Gln Gly Ser Asn Asn Ser Ala
Gln Ser Pro 115 120 125 Gln Thr Lys Thr Thr Pro Ala Pro Thr Thr Ser
Pro Met Thr Gln Asp 130 135 140 Pro Gln Glu Thr Ala Asn Ser Ser Lys
145 150 31912DNAMarburg virus 31atggccagtt ccagcaatta caacacatac
atgcaatact tgaacccccc tccttatgct 60gatcacggtg caaaccagtt gatcccggcg
gatcagctat caaatcagca gggtataact 120ccaaattacg tgggtgattt
aaacctagat gatcagttca aagggaatgt ctgccatgct 180ttcactttag
aggcaataat tgacatatct gcatataacg agcgaacagt caaaggcgtt
240ccggcatggc tgcctcttgg gattatgagc aattttgaat atcctttagc
tcatactgtg 300gccgcgttgc tcacaggcag ctatacaatc acccaattta
ctcacaacgg gcaaaaattc 360gtccgtgtta atcgacttgg tacaggaatc
ccagcacacc cactcagaat gttgcgtgaa 420ggaaatcaag cttttattca
gaatatggtg atccccagga atttttcaac taatcaattc 480acctacaatc
tcactaattt agtattgagt gtgcaaaaac ttcctgatga tgcctggcgc
540ccatccaagg acaaattaat tgggaacact atgcatcccg cagtctccat
ccacccgaat 600ctgccgccta ttgttctacc aacagtcaag aagcaggctt
atcgtcagca caaaaatccc 660aacaatggac cattgctggc catatctggc
atcctccatc aactgagggt cgaaaaagtc 720ccagagaaga cgagcctgtt
taggatctcg cttcctgccg acatgttctc agtaaaagag 780ggtatgatga
agaaaagggg agaaaattcc cccgtggttt attttcaagc acctgagaac
840ttccctttga atggcttcaa taacagacaa gttgtgctag cgtatgcgaa
tccaacgctc 900agtgccgttt ga 91232303PRTMarburg virus 32Met Ala Ser
Ser Ser Asn Tyr Asn Thr Tyr Met Gln Tyr Leu Asn Pro 1 5 10 15 Pro
Pro Tyr Ala Asp His Gly Ala Asn Gln Leu Ile Pro Ala Asp Gln 20 25
30 Leu Ser Asn Gln Gln Gly Ile Thr Pro Asn Tyr Val Gly Asp Leu Asn
35 40 45 Leu Asp Asp Gln Phe Lys Gly
Asn Val Cys His Ala Phe Thr Leu Glu 50 55 60 Ala Ile Ile Asp Ile
Ser Ala Tyr Asn Glu Arg Thr Val Lys Gly Val 65 70 75 80 Pro Ala Trp
Leu Pro Leu Gly Ile Met Ser Asn Phe Glu Tyr Pro Leu 85 90 95 Ala
His Thr Val Ala Ala Leu Leu Thr Gly Ser Tyr Thr Ile Thr Gln 100 105
110 Phe Thr His Asn Gly Gln Lys Phe Val Arg Val Asn Arg Leu Gly Thr
115 120 125 Gly Ile Pro Ala His Pro Leu Arg Met Leu Arg Glu Gly Asn
Gln Ala 130 135 140 Phe Ile Gln Asn Met Val Ile Pro Arg Asn Phe Ser
Thr Asn Gln Phe 145 150 155 160 Thr Tyr Asn Leu Thr Asn Leu Val Leu
Ser Val Gln Lys Leu Pro Asp 165 170 175 Asp Ala Trp Arg Pro Ser Lys
Asp Lys Leu Ile Gly Asn Thr Met His 180 185 190 Pro Ala Val Ser Ile
His Pro Asn Leu Pro Pro Ile Val Leu Pro Thr 195 200 205 Val Lys Lys
Gln Ala Tyr Arg Gln His Lys Asn Pro Asn Asn Gly Pro 210 215 220 Leu
Leu Ala Ile Ser Gly Ile Leu His Gln Leu Arg Val Glu Lys Val 225 230
235 240 Pro Glu Lys Thr Ser Leu Phe Arg Ile Ser Leu Pro Ala Asp Met
Phe 245 250 255 Ser Val Lys Glu Gly Met Met Lys Lys Arg Gly Glu Asn
Ser Pro Val 260 265 270 Val Tyr Phe Gln Ala Pro Glu Asn Phe Pro Leu
Asn Gly Phe Asn Asn 275 280 285 Arg Gln Val Val Leu Ala Tyr Ala Asn
Pro Thr Leu Ser Ala Val 290 295 300 332088DNAMarburg virus
33atggatttac acagtttgtt ggagttgggt acaaaaccca ctgcccctca tgttcgtaat
60aagaaagtga tattatttga cacaaatcat caggttagta tctgtaatca gataatagat
120gcaataaact cagggattga tcttggagat ctcctagaag ggggtttgct
gacgttgtgt 180gttgagcatt actataattc tgataaggat aaattcaaca
caagtcctat cgcgaagtac 240ttacgtgatg cgggctatga atttgatgtc
atcaagaatg cagatgcaac ccgctttctg 300gatgtgattc ctaatgaacc
tcattacagc cctttaattc tagcccttaa gacattggaa 360agtactgaat
ctcagagggg gagaattggg ctctttttat cattttgcag tcttttcctc
420ccaaaacttg tcgtcggaga ccgagctagt atcgaaaagg ctttaagaca
agtaacagtg 480catcaagaac aggggatcgt cacataccct aatcattggc
ttaccacagg ccacatgaaa 540gtaattttcg ggattttgag gtccagcttc
attttaaagt ttgtgttgat tcatcaagga 600gtaaatttgg tgacaggtca
tgatgcctat gacagtatca ttagtaattc agtaggtcaa 660actagattct
caggacttct tatcgtgaaa acagttctcg agttcatctt gcaaaaaact
720gattcagggg tgacactaca tcctttggtg cggacctcca aagtaaaaaa
tgaagttgct 780agtttcaagc aggcgttgag caacctagcc cgacatgggg
aatacgcacc atttgcacgg 840gttctgaatt tatcagggat taacaacctc
gaacatggac tctatcctca gctttcagca 900attgcgctgg gtgtggcaac
agcacacggc agtacattgg ctggtgtcaa tgttggcgaa 960caatatcaac
aactacgaga ggcggcacat gatgcggaag taaaactaca aaggcgacat
1020gaacatcagg aaattcaagc tattgccgag gatgacgagg aaaggaagat
attagaacaa 1080ttccaccttc agaaaactga aatcacacac agtcagacac
tagccgtcct cagccagaaa 1140cgagaaaaat tagctcgtct cgctgcagaa
attgaaaaca atattgtgga agatcaggga 1200tttaagcaat cacagaatcg
ggtgtcacag tcgtttttga atgaccctac acctgtggaa 1260gtaacggttc
aagccaggcc catgaatcga ccaactgctc tgcctccccc agttgacgac
1320aagattgagc atgaatctac agaagatagc tcttcttcaa gtagctttgt
tgacttgaat 1380gatccatttg cactgctgaa tgaggacgag gatactcttg
atgacagtgt catgatcccg 1440ggcacaacat cgagagaatt tcaagggatt
cctgaaccgc caagacaatc ccaagacctc 1500aataacagcc aaggaaagca
ggaagatgaa tccacaaatc cgattaagaa acagtttctg 1560agatatcaag
aattgcctcc tgttcaagag gatgatgaat cggaatacac aactgactct
1620caagaaagca tcgaccaacc aggatccgac aatgaacaag gagttgatct
tccacctcct 1680ccgttgtacg ctcaggaaaa aagacaggac ccaatacagc
acccagcagc aaaccctcag 1740gatcccttcg gcagtattgg tgatgtaaat
ggtgatatct tagaacctat aagatcacct 1800tcttcaccat ctgctcctca
ggaagacaca aggatgaggg aagcctatga attgtcgcct 1860gatttcacaa
atgatgagga taatcagcag aattggccac aaagagtggt gacaaagaag
1920ggtagaactt tcctttatcc taatgatctt ctgcaaacaa atcctccaga
gtcacttata 1980acagccctcg ttgaggaata ccaaaatcct gtctcagcta
aggagcttca agcagattgg 2040cccgacatgt catttgatga aaggagacat
gttgcgatga acttgtag 208834695PRTMarburg virus 34Met Asp Leu His Ser
Leu Leu Glu Leu Gly Thr Lys Pro Thr Ala Pro 1 5 10 15 His Val Arg
Asn Lys Lys Val Ile Leu Phe Asp Thr Asn His Gln Val 20 25 30 Ser
Ile Cys Asn Gln Ile Ile Asp Ala Ile Asn Ser Gly Ile Asp Leu 35 40
45 Gly Asp Leu Leu Glu Gly Gly Leu Leu Thr Leu Cys Val Glu His Tyr
50 55 60 Tyr Asn Ser Asp Lys Asp Lys Phe Asn Thr Ser Pro Ile Ala
Lys Tyr 65 70 75 80 Leu Arg Asp Ala Gly Tyr Glu Phe Asp Val Ile Lys
Asn Ala Asp Ala 85 90 95 Thr Arg Phe Leu Asp Val Ile Pro Asn Glu
Pro His Tyr Ser Pro Leu 100 105 110 Ile Leu Ala Leu Lys Thr Leu Glu
Ser Thr Glu Ser Gln Arg Gly Arg 115 120 125 Ile Gly Leu Phe Leu Ser
Phe Cys Ser Leu Phe Leu Pro Lys Leu Val 130 135 140 Val Gly Asp Arg
Ala Ser Ile Glu Lys Ala Leu Arg Gln Val Thr Val 145 150 155 160 His
Gln Glu Gln Gly Ile Val Thr Tyr Pro Asn His Trp Leu Thr Thr 165 170
175 Gly His Met Lys Val Ile Phe Gly Ile Leu Arg Ser Ser Phe Ile Leu
180 185 190 Lys Phe Val Leu Ile His Gln Gly Val Asn Leu Val Thr Gly
His Asp 195 200 205 Ala Tyr Asp Ser Ile Ile Ser Asn Ser Val Gly Gln
Thr Arg Phe Ser 210 215 220 Gly Leu Leu Ile Val Lys Thr Val Leu Glu
Phe Ile Leu Gln Lys Thr 225 230 235 240 Asp Ser Gly Val Thr Leu His
Pro Leu Val Arg Thr Ser Lys Val Lys 245 250 255 Asn Glu Val Ala Ser
Phe Lys Gln Ala Leu Ser Asn Leu Ala Arg His 260 265 270 Gly Glu Tyr
Ala Pro Phe Ala Arg Val Leu Asn Leu Ser Gly Ile Asn 275 280 285 Asn
Leu Glu His Gly Leu Tyr Pro Gln Leu Ser Ala Ile Ala Leu Gly 290 295
300 Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val Gly Glu
305 310 315 320 Gln Tyr Gln Gln Leu Arg Glu Ala Ala His Asp Ala Glu
Val Lys Leu 325 330 335 Gln Arg Arg His Glu His Gln Glu Ile Gln Ala
Ile Ala Glu Asp Asp 340 345 350 Glu Glu Arg Lys Ile Leu Glu Gln Phe
His Leu Gln Lys Thr Glu Ile 355 360 365 Thr His Ser Gln Thr Leu Ala
Val Leu Ser Gln Lys Arg Glu Lys Leu 370 375 380 Ala Arg Leu Ala Ala
Glu Ile Glu Asn Asn Ile Val Glu Asp Gln Gly 385 390 395 400 Phe Lys
Gln Ser Gln Asn Arg Val Ser Gln Ser Phe Leu Asn Asp Pro 405 410 415
Thr Pro Val Glu Val Thr Val Gln Ala Arg Pro Met Asn Arg Pro Thr 420
425 430 Ala Leu Pro Pro Pro Val Asp Asp Lys Ile Glu His Glu Ser Thr
Glu 435 440 445 Asp Ser Ser Ser Ser Ser Ser Phe Val Asp Leu Asn Asp
Pro Phe Ala 450 455 460 Leu Leu Asn Glu Asp Glu Asp Thr Leu Asp Asp
Ser Val Met Ile Pro 465 470 475 480 Gly Thr Thr Ser Arg Glu Phe Gln
Gly Ile Pro Glu Pro Pro Arg Gln 485 490 495 Ser Gln Asp Leu Asn Asn
Ser Gln Gly Lys Gln Glu Asp Glu Ser Thr 500 505 510 Asn Pro Ile Lys
Lys Gln Phe Leu Arg Tyr Gln Glu Leu Pro Pro Val 515 520 525 Gln Glu
Asp Asp Glu Ser Glu Tyr Thr Thr Asp Ser Gln Glu Ser Ile 530 535 540
Asp Gln Pro Gly Ser Asp Asn Glu Gln Gly Val Asp Leu Pro Pro Pro 545
550 555 560 Pro Leu Tyr Ala Gln Glu Lys Arg Gln Asp Pro Ile Gln His
Pro Ala 565 570 575 Ala Asn Pro Gln Asp Pro Phe Gly Ser Ile Gly Asp
Val Asn Gly Asp 580 585 590 Ile Leu Glu Pro Ile Arg Ser Pro Ser Ser
Pro Ser Ala Pro Gln Glu 595 600 605 Asp Thr Arg Met Arg Glu Ala Tyr
Glu Leu Ser Pro Asp Phe Thr Asn 610 615 620 Asp Glu Asp Asn Gln Gln
Asn Trp Pro Gln Arg Val Val Thr Lys Lys 625 630 635 640 Gly Arg Thr
Phe Leu Tyr Pro Asn Asp Leu Leu Gln Thr Asn Pro Pro 645 650 655 Glu
Ser Leu Ile Thr Ala Leu Val Glu Glu Tyr Gln Asn Pro Val Ser 660 665
670 Ala Lys Glu Leu Gln Ala Asp Trp Pro Asp Met Ser Phe Asp Glu Arg
675 680 685 Arg His Val Ala Met Asn Leu 690 695 35657DNAMarburg
virus 35gggctttcat caacaatgcc acccactccc tcaccacaac caagcacgcc
acagcaagga 60ggaaacaaca caaaccattc ccaagatgct gtgactgaac ttgacaaaaa
taacacaact 120gcacaaccgt ccatgccccc tcataacact accacaatct
ctactaacaa cacctccaaa 180cacaacttca gcactctctc tgcaccatta
caaaacacca ccaatgacaa cacacagagc 240acaatcactg aaaatgagca
aaccagtgcc ccctcgataa caaccctgcc tccaacggga 300aatcccacca
cagcaaagag caccagcagc aaaaaaggcc ccgccacaac ggcaccaaac
360acgacaaatg agcatttcac cagtcctccc cccaccccca gctcgactgc
acaacatctt 420gtatatttca gaagaaagcg aagtatcctc tggagggaag
gcgacatgtt cccttttctg 480gatgggttaa taaatgctcc aattgatttt
gacccagttc caaatacaaa aacaatcttt 540gatgaatcct ctagttctgg
tgcctcggct gaggaagatc aacatgcctc ccccaatatt 600agtttaactt
tatcttattt tcctaatata aatgagaaca ctgcctactc ttagtag
65736217PRTMarburg virus 36Gly Leu Ser Ser Thr Met Pro Pro Thr Pro
Ser Pro Gln Pro Ser Thr 1 5 10 15 Pro Gln Gln Gly Gly Asn Asn Thr
Asn His Ser Gln Asp Ala Val Thr 20 25 30 Glu Leu Asp Lys Asn Asn
Thr Thr Ala Gln Pro Ser Met Pro Pro His 35 40 45 Asn Thr Thr Thr
Ile Ser Thr Asn Asn Thr Ser Lys His Asn Phe Ser 50 55 60 Thr Leu
Ser Ala Pro Leu Gln Asn Thr Thr Asn Asp Asn Thr Gln Ser 65 70 75 80
Thr Ile Thr Glu Asn Glu Gln Thr Ser Ala Pro Ser Ile Thr Thr Leu 85
90 95 Pro Pro Thr Gly Asn Pro Thr Thr Ala Lys Ser Thr Ser Ser Lys
Lys 100 105 110 Gly Pro Ala Thr Thr Ala Pro Asn Thr Thr Asn Glu His
Phe Thr Ser 115 120 125 Pro Pro Pro Thr Pro Ser Ser Thr Ala Gln His
Leu Val Tyr Phe Arg 130 135 140 Arg Lys Arg Ser Ile Leu Trp Arg Glu
Gly Asp Met Phe Pro Phe Leu 145 150 155 160 Asp Gly Leu Ile Asn Ala
Pro Ile Asp Phe Asp Pro Val Pro Asn Thr 165 170 175 Lys Thr Ile Phe
Asp Glu Ser Ser Ser Ser Gly Ala Ser Ala Glu Glu 180 185 190 Asp Gln
His Ala Ser Pro Asn Ile Ser Leu Thr Leu Ser Tyr Phe Pro 195 200 205
Asn Ile Asn Glu Asn Thr Ala Tyr Ser 210 215
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