Human Ebola Virus Species and Compositions and Methods Thereof

Abstract

Compositions and methods including and related to the Ebola Bundibugyo virus (EboBun) are provided. Compositions are provided that are operable as immunogens to elicit and immune response or protection from EboBun challenge in a subject such as a primate. Inventive methods are directed to detection and treatment of EboBun infection.

Description

RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application 61/108,175 filed 24 Oct. 2008; the contents of which are hereby incorporated by reference.

DEPOSIT STATEMENT

[0002] The invention provides the isolated human Ebola (hEbola) viruses denoted as Bundibugyo (EboBun) deposited with the Centers for Disease Control and Prevention ("CDC"; Atlanta, Ga., United States of America) on Nov. 26, 2007 and accorded an accession number 200706291. This deposit was not made to an International Depository Authority (IDA) as established under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and is a non-Budapest treaty deposit. The deposited organism is not acceptable by American Type Culture Collection (ATCC), Manassas, Va., an International Depository Authority (IDA) as established under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Samples of the stated Deposit Accession No. 200706291 will be made available to approved facilities for thirty years from the date of deposit, and for the lifetime of the patent issuing from, or claiming priority to this application.

FIELD OF THE INVENTION

[0003] The invention is related to compositions and methods directed to a novel species of human Ebola (hEbola) virus.

BACKGROUND OF THE INVENTION

[0004] The family Filoviridae consists of two genera, Marburgvirus and Ebolavirus, which have likely evolved from a common ancestor¹. The genus Ebolavirus includes four species: Zaire, Sudan, Reston and Cote d'Ivoire (Ivory Coast) ebolaviruses, which have, with the exception of Reston and Cote d'Ivoire ebolaviruses, been associated with large hemorrhagic fever (HF) outbreaks in Africa with high case fatality (53-90%)².

[0005] Viruses of each species have genomes that are at least 30-40% divergent from one another, a level of diversity that presumably reflects differences in the ecological niche they occupy and in their evolutionary history. Identification of the natural reservoir of ebolaviruses remains somewhat elusive, although recent PCR and antibody data suggest that three species of arboreal fruit bats may be carriers of Zaire ebolavirus³. No data has yet been published to suggest reservoirs for the Sudan, Reston and Cote d'Ivoire ebolavirus species. However, a cave-dwelling fruit bat has been recently implicated as a natural host for marburgvirus⁴, 5, supporting the hypothesis that different bat species may be the reservoir hosts for the various filoviruses.

[0006] Filovirus outbreaks are sporadic, sometimes interspersed by years or even decades of no apparent disease activity. The last new species of ebolavirus was discovered 14 years ago (1994), in Cote d'Ivoire (Ivory Coast), and involved a single non-fatal case, a veterinarian who performed an autopsy on an infected chimpanzee found in the Tai Forest⁶. No further disease reports have been associated with Cote d'Ivoire ebolavirus, in contrast to Zaire and Sudan ebolaviruses which have each caused multiple large outbreaks over the same time period.

[0007] In late November 2007, HF cases were reported in the townships of Bundibugyo and Kikyo in Bundibugyo District, Western Uganda. The outbreak continued through January 2008, and resulted in approximately 149 cases and 37 deaths². Laboratory investigation of the initial 29 suspect-case blood specimens by classic methods (antigen capture, IgM and IgG ELISA) and a recently developed random-primed pyrosequencing approach identified this to be an Ebola HF outbreak associated with a new discovered ebolavirus species. These specimens were negative when initially tested with highly sensitive real-time RT-PCR assays specific for all known Zaire and Sudan ebolaviruses and Marburg viruses. This new species is referred to herein as "the Bundibugyo species", abbreviated "EboBun".

[0008] Accordingly, compositions and methods directed to the new Ebola virus species are described herein and the most closely related Ebola Ivory Coast species, which compositions and methods are useful for diagnosis and prevention of human Ebola virus infection; including related vaccine development, and prevention of hemorrhagic fever in a human population.

SUMMARY OF THE INVENTION

[0009] The present invention is based upon the isolation and identification of a new human Ebola virus species, EboBun. EboBun was isolated from the patients suffering from hemorrhagic fever in a recent outbreak in Uganda. The isolated virus is a member of the Filoviridae family, a family of negative sense RNA viruses. Accordingly, the invention relates to the isolated EboBun virus that morphologically and phylogenetically relates to known members filoviridae.

[0010] In one aspect, the invention provides the isolated EboBun virus deposited with the Centers for Disease Control and Prevention ("CDC"; Atlanta, Ga., United States of America) on Nov. 26, 2007 and accorded an accession number 200706291, as stated in the paragraph entitled "DEPOSIT STATEMENT" supra.

[0011] In another aspect, the invention provides an isolated hEbola EboBun virus comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence set forth in SEQ ID NO: 1; b) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 1 under stringent conditions; and c) a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the SEQ ID NO: 1. In another aspect, the invention provides the complete genomic sequence of the hEbola virus EboBun.

[0012] In a related aspect, the invention provides nucleic acid molecules isolated from EboBun, or fragments thereof.

[0013] In another aspect, the invention provides proteins or polypeptides that are isolated from the EboBun, including viral proteins isolated from cells infected with the virus but not present in comparable uninfected cells; or fragments thereof. In one embodiment of the present invention, the amino acid sequences of the proteins or polypeptides are set forth in SEQ ID NOS: 2-9 and 59, or fragments thereof.

[0014] In a related aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule of the inventive hEbola EboIC (Sequence ID No. 10) virus described above.

[0015] In another aspect, the invention provides an isolated hEbola EboIC virus comprising a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence set forth in SEQ ID NO: 10; b) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 10 under stringent conditions; and c) a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the SEQ ID NO: 10. In another aspect, the invention provides the complete genomic sequence of the hEbola virus EboIC.

[0016] In a related aspect, the invention provides nucleic acid molecules isolated from EboIC, or fragments thereof.

[0017] In another aspect, the invention provides proteins or polypeptides that are isolated from the EboIC, including viral proteins isolated from cells infected with the virus but not present in comparable uninfected cells; or fragments thereof. In one embodiment of the present invention, the amino acid sequences of the proteins or polypeptides are set forth in SEQ ID NOs: 11-19, or fragments thereof.

[0018] In a related aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule of the inventive hEbola EboIC virus described above.

[0019] In other aspects, the invention relates to the use of the isolated hEbola virus for diagnostic and therapeutic methods based on EbBun, EboIC, or a combination thereof. In one embodiment, the invention provides a method of detecting in a biological sample an antibody immunospecific for the genus of West Afrin Ebola Species constituting hEbola EbBun and EboIC virus using at least one the inventive isolated hEbola virus described herein, or any of the inventive proteins or polypeptides as described herein. In another specific embodiment, the invention provides a method of screening for an antibody which immunospecifically binds and neutralizes hEbola EboBun. Such an antibody is useful for a passive immunization or immunotherapy of a subject infected with hEbola.

[0020] In another aspect, the invention provides an isolated antibody or an antigen-binding fragment thereof which immunospecifically binds to the hEbola virus of the invention described above.

[0021] In other aspects, the invention provides methods for detecting the presence, activity or expression of the Glade of Bundibungyo-Ivory Coast hEbola virus in a biological material, such as cells, blood, saliva, urine, feces and so forth; and specifically at least one of EbBun or EboIC.

[0022] In a related aspect, the invention provides a method for detecting the presence of the inventive hEbola virus described above in a biological sample, the method includes (a) contacting the sample with an agent that selectively binds to a West African hEbola virus; and (b) detecting whether the compound binds to the West African hEbola virus in the sample.

[0023] In another aspect, the invention provides a method for detecting the presence of the inventive polypeptide described above, in a biological sample, said method includes (a) contacting the biological sample with an agent that selectively binds to the polypeptide; and (b) detecting whether the agent binds to the polypeptide in the sample. In another aspect, the invention provides a method for detecting the presence of a first nucleic acid molecule derived from the inventive hEbola virus described above in a biological sample, the method comprising: (a) contacting the biological sample with an agent that selectively binds to the polypeptide; and (b) detecting whether the agent binds to the polypeptide in the sample.

[0024] In another aspect, the invention provides a method for propagating the hEbola virus in host cells comprising infecting the host cells with the inventive isolated hEbola virus described above, culturing the host cells to allow the virus to multiply, and harvesting the resulting virions. Also provided by the present invention are host cells infected with the inventive hEbola virus described above.

[0025] In another aspect, the invention provides a method of detecting in a biological sample the presence of an antibody that immunospecifically binds hEbola virus, the method comprising: (a) contacting the biological sample with the inventive host cell host described above; and (b) detecting the antibody bound to the cell.

[0026] In another aspect, the invention provides vaccine preparations, comprising the inventive hEbola virus, including recombinant and chimeric forms of the virus, nucleic acid molecules comprised by the virus, or protein subunits of the virus. The invention also provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of the inventive hEbola virus described above, and a pharmaceutically acceptable carrier. In one embodiment, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the inventive hEbola virus described above, or a subunit thereof; and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a complement thereof, and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of inventive the nucleotide sequences as described above, or a complement thereof, and a pharmaceutically acceptable carrier.

[0027] In a related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of the inventive hEbola virus described above, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a protein extract of the inventive hEbola virus described above or a subunit thereof, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a complement thereof, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising the inventive nucleotide sequence as described above or a complement thereof, and a pharmaceutically acceptable carrier. In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of any of the inventive polypeptides described above.

[0028] In another aspect, the present invention provides pharmaceutical compositions comprising antiviral agents of the present invention and a pharmaceutically acceptable carrier. In a specific embodiment, the antiviral agent of the invention is an antibody that immunospecifically binds hEbola virus or any hEbola epitope. In another specific embodiment, the antiviral agent is a polypeptide or protein of the present invention or nucleic acid molecule of the invention.

[0029] In a related aspect, the invention provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of an anti-hEbola EboBun agent and a pharmaceutically acceptable carrier.

[0030] The invention also provides kits containing compositions and formulations of the present invention. Thus, in another aspect, the invention provides a kit comprising a container containing the inventive immunogenic formulation described above. In another aspect, the invention provides a kit comprising a container containing the inventive vaccine formulation described above. In another, the invention provides a kit comprising a container containing the inventive pharmaceutical composition described above. In another, the invention provides a kit comprising a container containing the inventive vaccine formulation described above. In another, the invention provides a method for identifying a subject infected with the inventive hEbola virus described above, comprising: (a) obtaining total RNA from a biological sample obtained from the subject; (b) reverse transcribing the total RNA to obtain cDNA; and (c) amplifying the cDNA using a set of primers derived from a nucleotide sequence of the inventive hEbola virus described above.

[0031] The invention further relates to the use of the sequence information of the isolated virus for diagnostic and therapeutic methods.

[0032] In another aspect, the present invention provides methods for screening antiviral agents that inhibit the infectivity or replication of hEbola virus or variants thereof.

[0033] The invention further provides methods of preparing recombinant or chimeric forms of hEbola.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 represents a Phylogenetic tree comparing full-length genomes of Ebolavirus and Marburg virus by Bayesian analysis;

[0035] FIG. 2 represents an alignment of genomes of novel hEbola EboBun (SEQ ID NO: 1) referred to below as "Ebola Bundibugyo" or "EboBun", and hEbola Zaire (SEQ ID NO: 20); referred to below as "Ebola Zaire '76" or "EboZ" and hEbola Ivory Coast (SEQ ID NO: 10) also referred to below as "EboIC".

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] It is to be understood that the present invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0037] Due to the sequence divergence of EboBun relative to all previously recognized ebolaviruses, the present invention has utility in design of diagnostic assays to monitor Ebola HF disease in humans and animals, and develop effective antivirals and vaccines.

[0038] The EboBun virus of the present invention is genetically distinct, differing by more than 30% at the genome level from all other known ebolavirus species. The unique nature of this virus created challenges for traditional filovirus molecular based diagnostic assays and genome sequencing approaches. Instead, over 70% of the virus genome was sequenced using a recently developed random-primed pyrosequencing approach which allowed the rapid development of molecular detection assay which were deployed in the disease outbreak response. This random-primed pyrosequencing draft sequence allowed faster completion of the whole genome sequence using traditional primer walking approach and confirmation that the EboBun virus represented a new ebolavirus species.

Definitions

[0039] The definitions herein provided are operative throughout the entire description of the invention set forth herein, including the Summary of the Invention.

[0040] The term "an antibody or an antibody fragment that immunospecifically binds a polypeptide of the invention" as used herein refers to an antibody or a fragment thereof that immunospecifically binds to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 (EboBun), or a fragment thereof, and does not non-specifically bind to other polypeptides. An antibody or a fragment thereof that immunospecifically binds to the polypeptide of the invention may cross-react with other antigens. Preferably, an antibody or a fragment thereof that immunospecifically binds to a polypeptide of the invention does not cross-react with other antigens. An antibody or a fragment thereof that immunospecifically binds to the polypeptide of the invention can be identified by, for example, immunoassays or other techniques known to those skilled in the art, or otherwise as described herein.

[0041] An "isolated" or "purified" peptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a polypeptide/protein in which the polypeptide/protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a polypeptide/protein that is substantially free of cellular material includes preparations of the polypeptide/protein having less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry weight) of contaminating protein. When the polypeptide/protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation.

[0042] When polypeptide/protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the polypeptide/protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than polypeptide/protein fragment of interest. In a preferred embodiment of the present invention, polypeptides/proteins are isolated or purified.

[0043] An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a preferred embodiment of the invention, nucleic acid molecules encoding polypeptides/proteins of the invention are isolated or purified. The term "isolated" nucleic acid molecule does not include a nucleic acid that is a member of a library that has not been purified away from other library clones containing other nucleic acid molecules.

[0044] The term "portion" or "fragment" as used herein includes the specified fragment lengths, and all integers in between, inclusive of the specified end points in a specified range, and inclusive of any length up to the full length of a protein, polypeptide, or nucleic acid.

[0045] The term "having a biological activity of the protein" or "having biological activities of the polypeptides of the invention" refers to the characteristics of the polypeptides or proteins having a common biological activity, similar or identical structural domain, and/or having sufficient amino acid identity to the polypeptide encoded by the nucleotide sequence of SEQ ID NO: 1 (EboBun). Such common biological activities of the polypeptides of the invention include antigenicity and immunogenicity.

[0046] The term "under stringent condition" refers to hybridization and washing conditions under which nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to each other remain hybridized to each other. Such hybridization conditions are described in, for example but not limited to, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6; Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., NY (1986), pp. 75-78, and 84-87; and Molecular Cloning, Cold Spring Harbor Laboratory, NY (1982), pp. 387-389, and are well known to those skilled in the art. A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC), 0.5% SDS at about 68° C. followed by one or more washes in 2×SSC, 0.5% SDS at room temperature. Another preferred, non-limiting example of stringent hybridization conditions is hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at about 50-65° C.

[0047] The term "variant" as used herein refers either to a naturally occurring genetic mutant of hEbola EboBun, or hEbola EboIC, or a recombinantly prepared variation of these hEbola species, each of which contain one or more mutations in its genome compared to the hEbola of SEQ ID NO: 1 or 10. The term "variant" may also refer either to a naturally occurring variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion.

[0048] "Homology" refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

[0049] The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

[0050] Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison, Wis.). CLUSTAL V is described in Higgins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins, D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The "weighted" residue weight table is selected as the default.

[0051] Alternatively, a suite of commonly used and freely available sequence comparison algorithms which can be used is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, Md., and on the NCBI World Wide Web site available on the Internet. The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called "BLAST 2 Sequences" that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively on the Internet via the NCBI World Wide Web site as well. The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21, 2000) set at default parameters. Such default parameters may be, for example: Matrix:BLOSUM62; Reward for match: 1; Penalty for mismatch: -2; Open Gap: 5 and Extension Gap: 2 penalties; Gap×drop-off: 50; Expect: 10; Word Size: 11; Filter: on.

[0052] Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or sequence listing, may be used to describe a length over which percentage identity may be measured.

[0053] The phrases "percent identity" and "% identity", as applied to polypeptide sequences, refer to the percentage of identical residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. The phrases "percent similarity" and "% similarity", as applied to polypeptide sequences, refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences aligned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences.

[0054] Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table.

[0055] Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21, 2000) with blastp set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap×drop-off: 50; Expect: 10; Word Size: 3; Filter: on.

[0056] Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or sequence listing, may be used to describe a length over which percentage identity may be measured.

[0057] The term "agent" encompasses any chemical, biochemical, or biological molecule; such as small molecules, proteins, polypeptides, antibodies, nucleic acid molecules including DNA or RNA, and the like.

Methods and Compositions Related to the Inventive hEbola

[0058] The present invention is based upon the isolation and identification of a new human Ebola virus species, EboBun and the sequencing of the only other known West African Ebola species EboIC. EboBun was isolated from the patients suffering from hemorrhagic fever in a recent outbreak in Uganda. The isolated virus is a member of the Filoviridae family, a family of negative sense RNA viruses. Accordingly, the invention relates to the isolated EboBun or EBOIC virus that morphologically and phylogenetically relates to known members filoviridae.

[0059] In another aspect, the invention provides an isolated hEbola virus including a nucleic acid molecule with a nucleotide sequence that is preferably: a) a nucleotide sequence set forth in SEQ ID NO: 1; b) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 1 under stringent conditions; or c) a nucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the SEQ ID NO: 1. In one embodiment of the present invention, the hEbola virus is killed. In another, the virus is attenuated. In another, the infectivity of the attenuated hEbola virus is reduced. In another, the infectivity is reduced by at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 10,000-fold. In another, the replication ability of the attenuated hEbola virus is reduced. In another, the replication ability of the attenuated virus is educed by at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, or 10,000-fold. In another, the protein synthesis ability of the attenuated virus is reduced. In another, the protein synthesis ability is reduced by at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, or 10,000-fold. In another, the assembling ability of the attenuated hEbola virus is reduced. In another, the assembling ability of the attenuated virus is reduced by at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, or 10,000-fold. In another, the cytopathic effect of the attenuated hEbola virus is reduced. In another, the cytopathic effect is reduced by at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, or 10,000-fold.

[0060] In another aspect, the invention provides the complete genomic sequence of the hEbola virus EboBun or EboIC. In a specific embodiment, the virus includes a nucleotide sequence of SEQ ID NOs: 1 or 10, respectively.

[0061] In a related aspect, the invention provides nucleic acid molecules isolated from EboBun, EboIC, or fragments thereof. In one embodiment of the present invention, the isolated nucleic acid molecule includes the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof. In another, the nucleic acid molecule includes a nucleotide sequence having at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4600, 4700, 4800, or 4900 contiguous nucleotides of the nucleotide sequence of SEQ ID NO: 1, or a complement thereof; with the proviso that the nucleotide sequence is not comprised by the nucleotide sequence set forth in SEQ ID NO: 20 (Ebola Zaire nucleotide sequence); or at least 5000, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, or 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof. In another embodiment, the isolated nucleic acid molecule includes a nucleotide sequence that encodes the EboBun amino acid sequence of SEQ ID NOs: 2-9 or 59, the EboIC amino acid sequence of SEQ ID NOs: 11-19, or a complement of the nucleotide sequence that encodes the EboBun amino acid sequences of SEQ ID NOs: 2-9 or 59 or the EboIC amino acid sequences of SEQ ID NOs: 11-19. In another, the isolated nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs: 1 or 10 or a complement thereof, wherein the nucleic acid molecule encodes an amino acid sequence which has a biological activity exhibited by a polypeptide encoded by the nucleotide sequence of SEQ ID NOs: 1 or 10. In another, nucleic acid molecule is RNA. In another, nucleic acid molecule is DNA.

[0062] In another aspect, the invention provides proteins or polypeptides that are isolated from the EboBun, including viral proteins isolated from cells infected with the virus but not present in comparable uninfected cells. In one embodiment of the present invention, the amino acid sequences of the proteins or polypeptides are set forth in SEQ ID NOs: 2-9, 59, or 11-19, or fragments thereof. In one embodiment, polypeptides or proteins of the present invention have a biological activity of the protein (including antigenicity and/or immunogenicity) encoded by the sequence of SEQ ID NOs: 1 or 10. In another, the polypeptides or the proteins of the present invention have a biological activity of at least one protein having the amino acid sequence (including antigenicity and/or immunogenicity) set forth in SEQ ID NOS: 2-9, 59, or 11-19, or a fragment thereof.

[0063] In a related aspect, the invention provides an isolated polypeptide encoded by the nucleic acid molecule of the invention described above. In one embodiment of the present invention, the isolated polypeptide includes the amino acid sequence selected from the group consisting of: a) an amino acid sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9; 11, 12, 13, 14, 15, 16, 17, 18 or 19; and b) an amino acid sequence that has 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to the amino acid sequence according to a). In another, the isolated polypeptide comprises the amino acid sequence having at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 210, 220, 230, 240 or 250 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 5 or 18 (VP24); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 210, 220, 230, 240, 250, 260, 270, 280 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 6 or 17 (VP30); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 310, or 320 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 8 or 13 (VP40); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 310, 320, 330, or 340 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 7 or 12 (VP35); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, or 370 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 4 or 15 (SGP); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 310, 320, 330, 340, 350, 360, or 370 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 59 or 16 (SSGP); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 450, 500, 550, 600, 610, 620, 630, 640, 650, 660, or 670 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 9 or 14 (GP); 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 450, 500, 550, 600, 650, 700, 710, 720, or 730 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 3 or 11 (NP); or 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2160, 2170, 2180, 2190, or 2200 contiguous amino acid residues of the amino acid sequence of SEQ ID NOs: 2 or 19 (L).

[0064] In other aspects, the invention relates to the use of an isolated West African hEbola virus for diagnostic and therapeutic methods. In one embodiment, the invention provides a method of detecting in a biological sample an antibody immunospecific for the hEbola virus using the inventive isolated hEbola virus described herein, or any of the inventive proteins or polypeptides as described herein. In another specific embodiment, the invention provides a method of screening for an antibody which immunospecifically binds and neutralizes hEbola EboBun or EboIC or a combination thereof. Such an antibody is useful for a passive immunization or immunotherapy of a subject infected with hEbola.

[0065] In another aspect, the invention provides an isolated antibody or an antigen-binding fragment thereof which immunospecifically binds to a West African genus hEbola virus of the invention described above, and illustratively including EboBun or EboIC. In one embodiment of the present invention, the isolated antibody or an antigen-binding fragment thereof neutralizes a West African genus hEbola virus. In another, the isolated antibody or an antigen-binding fragment thereof immunospecifically binds to the inventive polypeptide described above. The invention further provides antibodies that specifically bind a polypeptide of the invention encoded by the nucleotide sequence of SEQ ID NOs: 1 (EboBun) or 10 (EboIC), a fragment thereof, or encoded by a nucleic acid comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NOs: 1 (EboBun) or 10 (EboIC) and/or any hEbola EboBun epitope, having one or more biological activities of a polypeptide of the invention. These polypeptides include those shown in SEQ ID NOs: 2-9, 59, and 11-19. Such antibodies include, but are not limited to, polyclonal, monoclonal, bi-specific, multi-specific, human, humanized, chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, disulfide-linked Fvs, intrabodies and fragments containing either a VL or VH domain or even a complementary determining region (CDR) that specifically binds to a polypeptide of the invention.

[0066] In other aspects, the invention provides methods for detecting the presence, activity or expression of the hEbola virus of the invention in a biological material, such as cells, blood, saliva, urine, and so forth. The increased or decreased activity or expression of the hEbola virus in a sample relative to a control sample can be determined by contacting the biological material with an agent which can detect directly or indirectly the presence, activity or expression of the hEbola virus. In one embodiment of the present invention, the detecting agents are the antibodies or nucleic acid molecules of the present invention. Antibodies of the invention can also be used to treat hemorrhagic fever.

[0067] In a related aspect, the invention provides a method for detecting the presence of the inventive hEbola virus described above in a biological sample, the method comprising: (a) contacting the sample with an agent that selectively binds to the hEbola virus; and (b) detecting whether the compound binds to the hEbola virus in the sample. In one embodiment of the present invention, the biological sample is selected from the group consisting of cells; blood; serum; plasma; feces; rectal, vaginal and conjunctival swabs. In another, the agent that binds to the virus is an antibody. In another, the agent that binds to the virus is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a complement thereof. In another, the agent that binds to the virus is a nucleic acid molecule comprising a nucleotide sequence having at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, or 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof.

[0068] In another aspect, the invention provides a method for detecting the presence of the inventive polypeptide described above, in a biological sample, the method comprising: (a) contacting the biological sample with an agent that selectively binds to the polypeptide; and (b) detecting whether the agent binds to the polypeptide in the sample. In one embodiment of the present invention, the biological sample is selected from the group consisting of cells; blood; serum; plasma; feces; rectal, vaginal and conjunctival swabs. In another, the agent that binds to the polypeptide is an antibody or an antigen-binding fragment thereof.

[0069] In another aspect, the invention provides a method for detecting the presence of a first nucleic acid molecule derived from the inventive hEbola virus described above in a biological sample, the method includes (a) contacting the biological sample with an agent that selectively binds to the nucleic acid; and (b) detecting whether the agent binds to the nucleotide in the sample. In one embodiment of the present invention, the agent that binds to the first nucleic acid molecule is a second nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or a complement thereof. In another, the second nucleic acid molecule comprises at least 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 4600, 4700, 4800, 4900, 5000, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, or 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof.

[0070] In another aspect, the invention provides a method for propagating the hEbola virus in host cells comprising infecting the host cells with an inventive isolated West African hEbola virus described above, culturing the host cells to allow the virus to multiply, and harvesting the resulting virions. Also provided by the present invention are host cells infected with the inventive hEbola virus described above. In one embodiment of the present invention, the host cell is a primate cell.

[0071] In another aspect, the invention provides a method of detecting in a biological sample the presence of an antibody that immunospecifically binds hEbola virus, the method includes: (a) contacting the biological sample with the inventive host cell described above; and (b) detecting the antibody bound to the cell.

[0072] In another aspect, the invention provides vaccine preparations, including the inventive hEbola virus, including recombinant and chimeric forms of the virus, nucleic acid molecules comprised by the virus, or protein subunits of the virus. In one embodiment, the vaccine preparations of the present invention includes live but attenuated hEbola virus with or without pharmaceutically acceptable carriers, including adjuvants. In another, the vaccine preparations of the invention comprise an inactivated or killed hEbola EboBun virus, EboIC virus, or a combination thereof, with or without pharmaceutically acceptable carriers, including adjuvants. Such attenuated or inactivated viruses may be prepared by a series of passages of the virus through the host cells or by preparing recombinant or chimeric forms of virus. Accordingly, the present invention further provides methods of preparing recombinant or chimeric forms of the inventive hEbola viruses described herein.

[0073] In another specific embodiment, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of the inventive hEbola virus described above, and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the inventive hEbola virus described above, or a subunit thereof; and a pharmaceutically acceptable carrier. In another aspect, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof, and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of inventive the nucleotide sequences as described above, or a complement thereof, and a pharmaceutically acceptable carrier. In another aspect, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a protein extract of the inventive hEbola virus described above, or a subunit thereof; and a pharmaceutically acceptable carrier. In another aspect, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof, and a pharmaceutically acceptable carrier. In another, the invention provides a vaccine formulation comprising a therapeutically or prophylactically effective amount of a nucleic acid molecule comprising any of inventive the nucleotide sequences as described above, or a complement thereof, and a pharmaceutically acceptable carrier.

[0074] In yet another specific embodiment, the vaccine preparations of the present invention comprise a nucleic acid or fragment of the hEbola virus, e.g., the virus having Accession No. 200706291, or nucleic acid molecules having the sequence of SEQ ID NOs: 1 or 10, or a fragment thereof. In another, the vaccine preparations comprise a polypeptide of the invention encoded by the nucleotide sequence of SEQ ID NOs: 1 or 10 or a fragment thereof. In a specific embodiment, the vaccine preparations comprise polypeptides of the invention as shown in SEQ ID NOs: 2-9, 59, or 11-19, or encoded by the nucleotide sequence of SEQ ID NOs: 1 or 10, or a fragment thereof.

[0075] Furthermore, the present invention provides methods for treating, ameliorating, managing or preventing hemorrhagic fever by administering the vaccine preparations or antibodies of the present invention alone or in combination with adjuvants, or other pharmaceutically acceptable excipients. Furthermore, the present invention provides methods for treating, ameliorating, managing, or preventing hemorrhagic fever by administering the inventive compositions and formulations including the vaccine preparations or antibodies of the present invention alone or in combination with antivirals [e.g., amantadine, rimantadine, gancyclovir, acyclovir, ribavirin, penciclovir, oseltamivir, foscamet zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), stavudine (d4T), nevirapine, delavirdine, indinavir, ritonavir, vidarabine, nelfinavir, saquinavir, relenza, tamiflu, pleconaril, interferons, etc.], steroids and corticosteroids such as prednisone, cortisone, fluticasone and glucocorticoid, antibiotics, analgesics, bronchodilators, or other treatments for respiratory and/or viral infections.

[0076] In a related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of the inventive hEbola virus described above, and a pharmaceutically acceptable carrier.

[0077] In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a protein extract of the inventive hEbola virus described above or a subunit thereof, and a pharmaceutically acceptable carrier.

[0078] In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1, 10, a combination thereof, or a complement thereof, and a pharmaceutically acceptable carrier.

[0079] In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of a nucleic acid molecule comprising the inventive nucleotide sequence as described above or a complement thereof, and a pharmaceutically acceptable carrier.

[0080] In another related aspect, the invention provides an immunogenic formulation comprising an immunogenically effective amount of any of the inventive polypeptides described above.

[0081] In another aspect, the present invention provides pharmaceutical compositions comprising antiviral agents of the present invention and a pharmaceutically acceptable carrier. In a specific embodiment, the antiviral agent of the invention is an antibody that immunospecifically binds hEbola virus or any hEbola epitope. In another specific embodiment, the antiviral agent is a polypeptide or protein of the present invention or nucleic acid molecule of the invention.

[0082] In a related aspect, the invention provides a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of an anti-hEbola EboBun agent and a pharmaceutically acceptable carrier. In one embodiment of the present invention, the anti-hEbola EboBun agent is an antibody or an antigen-binding fragment thereof which immunospecifically binds to the hEbola virus of Deposit Accession No. 200706291, or polypeptides or protein derived therefrom. In another, the anti-hEbola agent is a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1, 10, a combination thereof, or a fragment thereof. In another, the anti-hEbola agent is a polypeptide encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOs: 1, 10, a combination thereof, or a fragment thereof having a biological activity of the polypeptide.

[0083] The invention also provides kits containing compositions and formulations of the present invention. Thus, in another aspect, the invention provides a kit comprising a container containing the inventive immunogenic formulation described above.

[0084] In another aspect, the invention provides a kit includes a container containing the inventive vaccine formulation described above.

[0085] In another aspect, the invention provides a kit including a container containing the inventive pharmaceutical composition described above.

[0086] In another aspect, the invention provides a kit including a container containing the inventive vaccine formulation described above.

[0087] In another aspect, the invention provides a method for identifying a subject infected with the inventive hEbola virus described above, including: (a) obtaining total RNA from a biological sample obtained from the subject; (b) reverse transcribing the total RNA to obtain cDNA; and (c) amplifying the cDNA using a set of primers derived from a nucleotide sequence of the inventive hEbola virus described above.

[0088] In one embodiment of the present invention, the set of primers are derived from the nucleotide sequence of the genome of the hEbola virus of Deposit Accession No. 200706291. In another, the set of primers are derived from the nucleotide sequence of SEQ ID NOs: 1 or 10 or any of the inventive nucleotide sequences as described above, or a complement thereof.

[0089] The invention further relates to the use of the sequence information of the isolated virus for diagnostic and therapeutic methods. In a specific embodiment, the invention provides nucleic acid molecules which are suitable for use as primers consisting of or including the nucleotide sequence of SEQ ID NOs: 1 or 10, or a complement thereof, or at least a portion of the nucleotide sequence thereof. In another specific embodiment, the invention provides nucleic acid molecules which are suitable for hybridization to the inventive hEbola nucleic acid; including, but not limited to PCR primers, Reverse Transcriptase primers, probes for Southern analysis or other nucleic acid hybridization analysis for the detection of hEbola nucleic acids, e.g., consisting of or including the nucleotide sequence of SEQ ID NOs: 1, 10 a combination thereof, a complement thereof, or a portion thereof. The invention further encompasses chimeric or recombinant viruses encoded in whole or in part by the nucleotide sequences.

[0090] In another aspect, the present invention provides methods for screening antiviral agents that inhibit the infectivity or replication of hEbola virus or variants thereof.

[0091] The invention further provides methods of preparing recombinant or chimeric forms of hEbola.

[0092] In another aspect, the invention provides vaccine preparations including the hEbola virus, including recombinant and chimeric forms of the virus, or subunits of the virus. The present invention encompasses recombinant or chimeric viruses encoded by viral vectors derived from the genome of the inventive hEbola virus described herein or natural variants thereof. In a specific embodiment, a recombinant virus is one derived from the hEbola virus of Deposit Accession No. 200706291. It is recognized that natural variants of the inventive hEbola viruses described herein comprise one or more mutations, including, but not limited to, point mutations, rearrangements, insertions, deletions etc., to the genomic sequence. It is recognized that the mutations may or may not result in a phenotypic change.

[0093] In another specific embodiment, a chimeric virus of the invention is a recombinant hEbola EboBun or EboIC virus which further comprises a heterologous nucleotide sequence. In accordance with the invention, a chimeric virus may be encoded by a nucleotide sequence in which heterologous nucleotide sequences have been added to the genome or in which endogenous or native nucleotide sequences have been replaced with heterologous nucleotide sequences.

[0094] According to the present invention, the chimeric viruses are encoded by the viral vectors of the invention which further comprise a heterologous nucleotide sequence. In accordance with the present invention a chimeric virus is encoded by a viral vector that may or may not include nucleic acids that are non-native to the viral genome. In accordance with the invention a chimeric virus is encoded by a viral vector to which heterologous nucleotide sequences have been added, inserted or substituted for native or non-native sequences. In accordance with the present invention, the chimeric virus may be encoded by nucleotide sequences derived from different species or variants of hEbola virus. In particular, the chimeric virus is encoded by nucleotide sequences that encode antigenic polypeptides derived from different species or variants of hEbola virus.

[0095] A chimeric virus may be of particular use for the generation of recombinant vaccines protecting against two or more viruses (Tao et al., J. Virol. 72, 2955-2961; Durbin et al., 2000, J. Virol. 74, 6821-6831; Skiadopoulos et al., 1998, J. Virol. 72, 1762-1768 (1998); Teng et al., 2000, J. Virol. 74, 9317-9321). For example, it can be envisaged that a virus vector derived from the hEbola virus expressing one or more proteins of variants of hEbola virus including hEbola EboBun, or vice versa, will protect a subject vaccinated with such vector against infections by both the native hEbola and the variant. Attenuated and replication-defective viruses may be of use for vaccination purposes with live vaccines as has been suggested for other viruses. (See, for example, PCT WO 02/057302, at pp. 6 and 23; and United States Patent Application Publication 2008/0069838 incorporated by reference herein).

[0096] In accordance with the present invention the heterologous sequence to be incorporated into the viral vectors encoding the recombinant or chimeric viruses of the invention include sequences obtained or derived from different species or variants of hEbola.

[0097] In certain embodiments, the chimeric or recombinant viruses of the invention are encoded by viral vectors derived from viral genomes wherein one or more sequences, intergenic regions, termini sequences, or portions or entire ORF have been substituted with a heterologous or non-native sequence. In certain embodiments of the invention, the chimeric viruses of the invention are encoded by viral vectors derived from viral genomes wherein one or more heterologous sequences have been inserted or added to the vector.

[0098] The selection of the viral vector may depend on the species of the subject that is to be treated or protected from a viral infection. If the subject is human, then an attenuated hEbola virus can be used to provide the antigenic sequences.

[0099] In accordance with the present invention, the viral vectors can be engineered to provide antigenic sequences which confer protection against infection by the inventive hEbola and natural variants thereof. The viral vectors may be engineered to provide one, two, three or more antigenic sequences. In accordance with the present invention the antigenic sequences may be derived from the same virus, from different species or variants of the same type of virus, or from different viruses.

[0100] The expression products and/or recombinant or chimeric virions obtained in accordance with the invention may advantageously be utilized in vaccine formulations. The expression products and chimeric virions of the present invention may be engineered to create vaccines against a broad range of pathogens, including viral and bacterial antigens, tumor antigens, allergen antigens, and auto antigens involved in autoimmune disorders. One way to achieve this goal involves modifying existing hEbola genes to contain foreign sequences in their respective external domains. Where the heterologous sequences are epitopes or antigens of pathogens, these chimeric viruses may be used to induce a protective immune response against the disease agent from which these determinants are derived. In particular, the chimeric virions of the present invention may be engineered to create vaccines for the protection of a subject from infections with hEbola virus and variants thereof.

[0101] Thus, the present invention further relates to the use of viral vectors and recombinant or chimeric viruses to formulate vaccines against a broad range of viruses and/or antigens. The present invention also encompasses recombinant viruses including a viral vector derived from the hEbola or variants thereof which contains sequences which result in a virus having a phenotype more suitable for use in vaccine formulations, e.g., attenuated phenotype or enhanced antigenicity. The mutations and modifications can be in coding regions, in intergenic regions and in the leader and trailer sequences of the virus.

[0102] The invention provides a host cell including a nucleic acid or a vector according to the invention. Plasmid or viral vectors containing the polymerase components of hEbola virus are generated in prokaryotic cells for the expression of the components in relevant cell types (bacteria, insect cells, eukaryotic cells). Plasmid or viral vectors containing full-length or partial copies of the hEbola genome will be generated in prokaryotic cells for the expression of viral nucleic acids in vitro or in vivo. The latter vectors optionally contain other viral sequences for the generation of chimeric viruses or chimeric virus proteins, optionally lack parts of the viral genome for the generation of replication defective virus, and optionally contain mutations, deletions or insertions for the generation of attenuated viruses. In addition, the present invention provides a host cell infected with hEbola virus of Deposit Accession No. 200706291,

[0103] Infectious copies of West African hEbola (being wild type, attenuated, replication-defective or chimeric) are optionally produced upon co-expression of the polymerase components according to the state-of-the-art technologies described above.

[0104] In addition, eukaryotic cells, transiently or stably expressing one or more full-length or partial hEbola proteins are optionally used. Such cells are preferably made by transfection (proteins or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors) and are useful for complementation of mentioned wild type, attenuated, replication-defective or chimeric viruses.

[0105] The viral vectors and chimeric viruses of the present invention optionally modulate a subject's immune system by stimulating a humoral immune response, a cellular immune response or by stimulating tolerance to an antigen. As used herein, a subject means: humans, primates, horses, cows, sheep, pigs, goats, dogs, cats, avian species and rodents.

Formulation of Vaccines and Antivirals

[0106] In a preferred embodiment, the invention provides a proteinaceous molecule or hEbola virus specific viral protein or functional fragment thereof encoded by a nucleic acid according to the invention. Useful proteinaceous molecules are for example derived from any of the genes or genomic fragments derivable from the virus according to the invention, preferably the GP, L, NP, sGP, VP24, VP30, VP35, and VP 40 proteins described herein. Such molecules, or antigenic fragments thereof, as provided herein, are for example useful in diagnostic methods or kits and in pharmaceutical compositions such as subunit vaccines. Particularly useful are polypeptides encoded by the nucleotide sequence of SEQ ID NOs: 1 or 10; or antigenic fragments thereof for inclusion as antigen or subunit immunogen, but inactivated whole virus can also be used. Particularly useful are also those proteinaceous substances that are encoded by recombinant nucleic acid fragments of the hEbola genome, of course preferred are those that are within the preferred bounds and metes of ORFs, in particular, for eliciting hEbola specific antibody or T cell responses, whether in vivo (e.g. for protective or therapeutic purposes or for providing diagnostic antibodies) or in vitro (e.g. by phage display technology or another technique useful for generating synthetic antibodies).

[0107] It is recognized that numerous variants, analogues, or homologues of EboBun polypeptides are within the scope of the present invention including amino acid substitutions, alterations, modifications, or other amino acid changes that increase, decrease, or do not alter the function or immunogenic propensity of the inventive immunogen or vaccine. Several post-translational modifications are similarly envisioned as within the scope of the present invention illustratively including incorporation of a non-naturally occurring amino acid(s), phosphorylation, glycosylation, sulfation, and addition of pendent groups such as biotynlation, fluorophores, lumiphores, radioactive groups, antigens, or other molecules.

[0108] Methods of expressing and purifying natural or recombinant peptides and proteins are well known in the art. Illustratively, peptides and proteins are recombinantly expressed in eukaryotic cells. Exemplary eukaryotic cells include yeast, HeLa cells, 293 cells, COS cells, Chinese hamster ovary cells (CHO), and many other cell types known in the art. Both eukaryotic and prokaryotic expression systems and cells are available illustratively from Invitrogen Corp., Carlsbad, Calif. It is appreciated that cell-free expression systems are similarly operable.

[0109] In a preferred embodiment an immunogenic polypeptide is a full length EboBun protein. Preferably, an immunogen is a full length EboBun protein of SEQ ID NOs: 2-9 or 59, or EboIC SEQ ID NOs: 11-19, or a fragment thereof as described herein. Preferably, an immunogen is has a minimum of 5 amino acids. As used herein an immunogen is preferably a polypeptide. In the context of an immunogenic polypeptide the terms immunogen, polypeptide, and antigen are used interchangeably.

[0110] Modifications and changes can be made in the structure of the inventive immunogens that are the subject of the application and still obtain a molecule having similar or improved characteristics as the wild-type sequence (e.g., a conservative amino acid substitution). For example, certain amino acids are optionally substituted for other amino acids in a sequence without appreciable loss of immunogenic activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like or improved properties. Optionally, a polypeptide is used that has less or more immunogenic activity compared to the wild-type sequence.

[0111] In making such changes, the hydropathic index of amino acids is preferably considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0112] It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent immunogen. In such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0113] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide and immunogen as set forth above. In particular, embodiments of the polypeptides and immunogens optionally include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.

[0114] The invention provides vaccine formulations for the prevention and treatment of infections with hEbola virus. In certain embodiments, the vaccine of the invention comprises recombinant and chimeric viruses of the hEbola virus. In certain embodiments, the virus is attenuated.

[0115] In another embodiment of this aspect of the invention, inactivated vaccine formulations are prepared using conventional techniques to "kill" the chimeric viruses. Inactivated vaccines are "dead" in the sense that their infectivity has been destroyed. Ideally, the infectivity of the virus is destroyed without affecting its immunogenicity. In order to prepare inactivated vaccines, the chimeric virus may be grown in cell culture or in the allantois of the chick embryo, purified by zonal ultracentrifugation, inactivated by formaldehyde or β-propiolactone, and pooled. The resulting vaccine is usually inoculated intramuscularly or intranasally.

[0116] Inactivated viruses are optionally formulated with a suitable adjuvant in order to enhance the immunological response. Such adjuvants illustratively include but are not limited to mineral gels, e.g., aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; peptides; oil emulsions; and potentially useful human adjuvants such as BCG and Corynebacterium parvum.

[0117] In another aspect, the present invention also provides DNA vaccine formulations including a nucleic acid or fragment of the inventive hEbola virus, e.g., the virus having Accession No. 200706291, or nucleic acid molecules having the sequence of SEQ ID NOs: 1 or 10, or a fragment thereof. In another specific embodiment, the DNA vaccine formulations of the present invention comprise a nucleic acid or fragment thereof encoding the antibodies which immunospecifically bind hEbola viruses. In DNA vaccine formulations, a vaccine DNA comprises a viral vector, such as that derived from the hEbola virus, bacterial plasmid, or other expression vector, bearing an insert including a nucleic acid molecule of the present invention operably linked to one or more control elements, thereby allowing expression of the vaccinating proteins encoded by the nucleic acid molecule in a vaccinated subject. Such vectors can be prepared by recombinant DNA technology as recombinant or chimeric viral vectors carrying a nucleic acid molecule of the present invention.

[0118] A nucleic acid as used herein refers to single- or double-stranded molecules which are optionally DNA, including the nucleotide bases A, T, C and G, or RNA, including the bases A, U (substitutes for T), C, and G. The nucleic acid may represent a coding strand or its complement. Nucleic acids are optionally identical in sequence to the sequence which is naturally occurring or include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids optionally include codons which represent conservative substitutions of amino acids as are well known in the art.

[0119] As used herein, the term "isolated nucleic acid" means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The isolation of nucleic acids is illustratively accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids. The nucleic acids of this invention are illustratively isolated from cells according to methods well known in the art for isolating nucleic acids. Alternatively, the nucleic acids of the present invention are optionally synthesized according to standard protocols well described in the literature for synthesizing nucleic acids. Modifications to the nucleic acids of the invention are also contemplated, provided that the essential structure and function of the peptide or polypeptide encoded by the nucleic acid are maintained.

[0120] The nucleic acid encoding the peptide or polypeptide of this invention is optionally part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations. Thus, the present invention further provides a recombinant nucleic acid construct including a nucleic acid encoding a polypeptide of this invention.

[0121] Generally, it may be more convenient to employ as the recombinant polynucleotide a cDNA version of the polynucleotide. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventor does not exclude the possibility of employing a genomic version of a particular gene where desired.

[0122] As used herein, the terms "engineered" and "recombinant" cells are synonymous with "host" cells and are intended to refer to a cell into which an exogenous DNA segment or gene, such as a cDNA or gene has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. A host cell is optionally a naturally occurring cell that is transformed with an exogenous DNA segment or gene or a cell that is not modified. A host cell preferably does not possess a naturally occurring gene encoding RSV G protein. Engineered cells are, thus, cells having a gene or genes introduced through the hand of man. Recombinant cells illustratively include those having an introduced cDNA or genomic DNA, and also include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.

[0123] To express a recombinant encoded polypeptide in accordance with the present invention one optionally prepares an expression vector that comprises a polynucleotide under the control of one or more promoters. To bring a coding sequence "under the control of" a promoter, one positions the 5' end of the translational initiation site of the reading frame generally between about 1 and 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the inserted DNA and promotes expression of the encoded recombinant protein. This is the meaning of "recombinant expression" in the context used here.

[0124] Many standard techniques are available to construct expression vectors containing the appropriate nucleic acids and transcriptional/translational control sequences in order to achieve protein or peptide expression in a variety of host-expression systems. Cell types available for expression include, but are not limited to, bacteria, such as E. coli and B. subtilis transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors.

[0125] Certain examples of prokaryotic hosts illustratively include E. coli strain RR1, E. coli LE392, E. coli B, E. coli 1776 (ATCC No. 31537) as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillus subtilis; and other enterobacteria such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species.

[0126] In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli is often transformed using pBR322, a plasmid derived from an E. coli species. Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid or phage may also contain, or be modified to contain, promoters that can be used by the microbial organism for expression of its own proteins.

[0127] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism are optionally used as transforming vectors in connection with these hosts. For example, the phage lambda is optionally utilized in making a recombinant phage vector that can be used to transform host cells, such as E. coli LE392.

[0128] Further useful vectors include pIN vectors and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. Other suitable fusion proteins are those with β-galactosidase, ubiquitin, or the like.

[0129] Promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling those of skill in the art to ligate them functionally with plasmid vectors.

[0130] For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used. This plasmid contains the trp1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0131] Suitable promoting sequences in yeast vectors illustratively include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also preferably ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.

[0132] Other suitable promoters, which have the additional advantage of transcription controlled by growth conditions, illustratively include the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.

[0133] In addition to microorganisms, cultures of cells derived from multicellular organisms are also operable as hosts. In principle, any such cell culture is operable, whether from vertebrate or invertebrate culture. In addition to mammalian cells, these include insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); and plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing one or more coding sequences.

[0134] In a useful insect system, Autographica californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The isolated nucleic acid coding sequences are cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of the coding sequences results in the inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., U.S. Pat. No. 4,215,051).

[0135] Examples of useful mammalian host cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN and MDCK cell lines. In addition, a host cell is preferably chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the encoded protein.

[0136] Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems are preferably chosen to ensure the correct modification and processing of the foreign protein expressed. Expression vectors for use in mammalian cells ordinarily include an origin of replication (as necessary), a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. The origin of replication is preferably provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

[0137] The promoters are optionally derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Further, it is also possible, and may be desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

[0138] A number of viral based expression systems are operable herein, for example, commonly used promoters are derived from polyoma, Adenovirus 2, Adenovirus 5, cytomegalovirus and Simian Virus 40 (SV40). The early and late promoters of SV40 virus are useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication. Smaller or larger SV40 fragments are also operable, particularly when there is included the approximately 250 bp sequence extending from the HindIII site toward the BglI site located in the viral origin of replication.

[0139] In cases where an adenovirus is used as an expression vector, the coding sequences are preferably ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene is then optionally inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing proteins in infected hosts.

[0140] Specific initiation signals may also be required for efficient translation of the claimed isolated nucleic acid coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this need and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons are optionally of a variety of origins, both natural and synthetic. The efficiency of expression is optionally enhanced by the inclusion of appropriate transcription enhancer elements or transcription terminators.

[0141] In eukaryotic expression, one will also typically desire to incorporate into the transcriptional unit an appropriate polyadenylation site if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.

[0142] For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express constructs encoding proteins are engineered. Rather than using expression vectors that contain viral origins of replication, host cells are preferably transformed with vectors controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched medium, and then are switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines.

[0143] A number of selection systems are illustratively used, including, but not limited, to the herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance is optionally used as the basis of selection for dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G-418; and hygro, which confers resistance to hygromycin. It is appreciated that numerous other selection systems are known in the art that are similarly operable in the present invention.

[0144] The nucleic acids encoding the peptides and polypeptides of this invention are optionally administered as nucleic acid vaccines. For the purposes of vaccine delivery, a nucleic acid encoding a peptide or polypeptide of this invention is preferably in an expression vector that includes viral nucleic acid including, but not limited to, vaccinia virus, adenovirus, retrovirus and/or adeno-associated virus nucleic acid. The nucleic acid or vector of this invention is optionally in a liposome or a delivery vehicle which can be taken up by a cell via receptor-mediated or other type of endocytosis. The nucleic acid vaccines of this invention are preferably in a pharmaceutically acceptable carrier or administered with an adjuvant. The nucleic acids encoding the peptides and polypeptides of this invention can also be administered to cells in vivo or ex vivo.

[0145] It is contemplated that the isolated nucleic acids of the disclosure are optionally "overexpressed", i.e., expressed in increased levels relative to its natural expression in cells of its indigenous organism, or even relative to the expression of other proteins in the recombinant host cell. Such overexpression is assessed by a variety of methods illustratively including radio-labeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or immunoblotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural in transfected cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.

[0146] Various heterologous vectors are described for DNA vaccinations against viral infections. For example, the vectors described in the following references, incorporated herein by reference, may be used to express hEbola sequences instead of the sequences of the viruses or other pathogens described; in particular, vectors described for hepatitis B virus (Michel, M. L. et al., 1995, DAN-mediated immunization to the hepatitis B surface antigen in mice: Aspects of the humoral response mimic hepatitis B viral infection in humans, Proc. Natl. Aca. Sci. USA 92:5307-5311; Davis, H. L. et al., 1993, DNA-based immunization induces continuous secretion of hepatitis B surface antigen and high levels of circulating antibody, Human Molec. Genetics 2:1847-1851), HIV virus (Wang, B. et al., 1993, Gene inoculation generates immune responses against human immunodeficiency virus type 1, Proc. Natl. Acad. Sci. USA 90:4156-4160; Lu, S. et al., 1996, Simian immunodeficiency virus DNA vaccine trial in Macques, J. Virol. 70:3978-3991; Letvin, N. L. et al., 1997, Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination, Proc Natl Acad Sci USA. 94(17):9378-83), and influenza viruses (Robinson, H L et al., 1993, Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA, Vaccine 11:957-960; Ulmer, J. B. et al., Heterologous protection against influenza by injection of DNA encoding a viral protein, Science 259:1745-1749), as well as bacterial infections, such as tuberculosis (Tascon, R. E. et al., 1996, Vaccination against tuberculosis by DNA injection, Nature Med. 2:888-892; Huygen, K. et al., 1996, Immunogenicity and protective efficacy of a tuberculosis DNA vaccine, Nature Med., 2:893-898), and parasitic infection, such as malaria (Sedegah, M., 1994, Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein, Proc. Natl. Acad. Sci. USA 91:9866-9870; Doolan, D. L. et al., 1996, Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD8+T cell-interferon δ, and nitric oxide-dependent immunity, J. Exper. Med., 1183:1739-1746).

[0147] Many methods are optionally used to introduce the vaccine formulations described above. These include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes. Alternatively, in a preferred embodiment the chimeric virus vaccine formulation is introduced via the natural route of infection of the pathogen for which the vaccine is designed. The DNA vaccines of the present invention are optionally administered in saline solutions by injections into muscle or skin using a syringe and needle (Wolff J. A. et al., 1990, Direct gene transfer into mouse muscle in vivo, Science 247:1465-1468; Raz, E., 1994, Intradermal gene immunization: The possible role of DNA uptake in the induction of cellular immunity to viruses, c. Natl. Acd. Sci. USA 91:9519-9523). Another way to administer DNA vaccines operable herein is called the "gene gun" method, whereby microscopic gold beads coated with the DNA molecules of interest is fired into cells (Tang, D. et al., 1992, Genetic immunization is a simple method for eliciting an immune response, Nature 356:152-154). For general reviews of the methods for DNA vaccines, see Robinson, H. L., 1999, DNA vaccines: basic mechanism and immune responses (Review), Int. J. Mol. Med. 4(5):549-555; Barber, B., 1997, Introduction: Emerging vaccine strategies, Seminars in Immunology 9(5):269-270; and Robinson, H. L. et al., 1997, DNA vaccines, Seminars in Immunology 9(5):271-283.

Attenuation of hEbola Virus or Variants Thereof

[0148] The hEbola virus or variants thereof of the invention are optionally genetically engineered to exhibit an attenuated phenotype. In particular, the viruses of the invention exhibit an attenuated phenotype in a subject to which the virus is administered as a vaccine. Attenuation can be achieved by any method known to a skilled artisan. Without being bound by theory, the attenuated phenotype of the viruses of the invention is caused, e.g., by using a virus that naturally does not replicate well in an intended host species, for example, by reduced replication of the viral genome, by reduced ability of the virus to infect a host cell, or by reduced ability of the viral proteins to assemble to an infectious viral particle relative to the wild type species of the virus.

[0149] The attenuated phenotypes of hEbola virus or variants thereof are optionally tested by any method known to the artisan. A candidate virus, for example, is optionally tested for its ability to infect a host or for the rate of replication in a cell culture system. In certain embodiments, growth curves at different temperatures are used to test the attenuated phenotype of the virus. For example, an attenuated virus is able to grow at 35° C., but not at 39° C. or 40° C. In certain embodiments, different cell lines are used to evaluate the attenuated phenotype of the virus. For example, an attenuated virus may only be able to grow in monkey cell lines but not the human cell lines, or the achievable virus titers in different cell lines are different for the attenuated virus. In certain embodiments, viral replication in the respiratory tract of a small animal model, including but not limited to, hamsters, cotton rats, mice and guinea pigs, is used to evaluate the attenuated phenotypes of the virus. In other embodiments, the immune response induced by the virus, including but not limited to, the antibody titers (e.g., assayed by plaque reduction neutralization assay or ELISA) is used to evaluate the attenuated phenotypes of the virus. In a specific embodiment, the plaque reduction neutralization assay or ELISA is carried out at a low dose. In certain embodiments, the ability of the hEbola virus to elicit pathological symptoms in an animal model is tested. A reduced ability of the virus to elicit pathological symptoms in an animal model system is indicative of its attenuated phenotype. In a specific embodiment, the candidate viruses are tested in a monkey model for nasal infection, indicated by mucus production.

[0150] The viruses of the invention are optionally attenuated such that one or more of the functional characteristics of the virus are impaired. In certain embodiments, attenuation is measured in comparison to the wild type species of the virus from which the attenuated virus is derived. In other embodiments, attenuation is determined by comparing the growth of an attenuated virus in different host systems. Thus, for a non-limiting example, hEbola virus or a variant thereof is attenuated when grown in a human host if the growth of the hEbola or variant thereof in the human host is reduced compared to the non-attenuated hEbola or variant thereof.

[0151] In certain embodiments, the attenuated virus of the invention is capable of infecting a host, is capable of replicating in a host such that infectious viral particles are produced. In comparison to the wild type species, however, the attenuated species grows to lower titers or grows more slowly. Any technique known to the skilled artisan can be used to determine the growth curve of the attenuated virus and compare it to the growth curve of the wild type virus.

[0152] In certain embodiments, the attenuated virus of the invention (e.g., a recombinant or chimeric hEbola) cannot replicate in human cells as well as the wild type virus (e.g., wild type hEbola) does. However, the attenuated virus can replicate well in a cell line that lacks interferon functions, such as Vero cells.

[0153] In other embodiments, the attenuated virus of the invention is capable of infecting a host, of replicating in the host, and of causing proteins of the virus of the invention to be inserted into the cytoplasmic membrane, but the attenuated virus does not cause the host to produce new infectious viral particles. In certain embodiments, the attenuated virus infects the host, replicates in the host, and causes viral proteins to be inserted in the cytoplasmic membrane of the host with the same efficiency as the wild type hEbola. In other embodiments, the ability of the attenuated virus to cause viral proteins to be inserted into the cytoplasmic membrane into the host cell is reduced compared to the wild type virus. In certain embodiments, the ability of the attenuated hEbola virus to replicate in the host is reduced compared to the wild type virus. Any technique known to the skilled artisan can be used to determine whether a virus is capable of infecting a mammalian cell, of replicating within the host, and of causing viral proteins to be inserted into the cytoplasmic membrane of the host.

[0154] In certain embodiments, the attenuated virus of the invention is capable of infecting a host. In contrast to the wild type hEbola, however, the attenuated hEbola cannot be replicated in the host. In a specific embodiment, the attenuated hEbola virus can infect a host and can cause the host to insert viral proteins in its cytoplasmic membranes, but the attenuated virus is incapable of being replicated in the host. Any method known to the skilled artisan can be used to test whether the attenuated hEbola has infected the host and has caused the host to insert viral proteins in its cytoplasmic membranes.

[0155] In certain embodiments, the ability of the attenuated virus to infect a host is reduced compared to the ability of the wild type virus to infect the same host. Any technique known to the skilled artisan can be used to determine whether a virus is capable of infecting a host.

[0156] In certain embodiments, mutations (e.g., missense mutations) are introduced into the genome of the virus, for example, into the sequence of SEQ ID NOs: 1 or 10, or to generate a virus with an attenuated phenotype. Mutations (e.g., missense mutations) can be introduced into the structural genes and/or regulatory genes of the hEbola. Mutations are optionally additions, substitutions, deletions, or combinations thereof. Such variant of hEbola can be screened for a predicted functionality, such as infectivity, replication ability, protein synthesis ability, assembling ability, as well as cytopathic effect in cell cultures. In a specific embodiment, the missense mutation is a cold-sensitive mutation. In another embodiment, the missense mutation is a heat-sensitive mutation. In another embodiment, the missense mutation prevents a normal processing or cleavage of the viral proteins.

[0157] In other embodiments, deletions are introduced into the genome of the hEbola virus, which result in the attenuation of the virus.

[0158] In certain embodiments, attenuation of the virus is achieved by replacing a gene of the wild type virus with a gene of a virus of a different species, of a different subgroup, or of a different variant. In another aspect, attenuation of the virus is achieved by replacing one or more specific domains of a protein of the wild type virus with domains derived from the corresponding protein of a virus of a different species. In certain other embodiments, attenuation of the virus is achieved by deleting one or more specific domains of a protein of the wild type virus.

[0159] When a live attenuated vaccine is used, its safety should also be considered. The vaccine preferably does not cause disease. Any techniques known in the art for improving vaccine safety are operable in the present invention. In addition to attenuation techniques, other techniques are optionally be used. One non-limiting example is to use a soluble heterologous gene that cannot be incorporated into the virion membrane. For example, a single copy of the soluble version of a viral transmembrane protein lacking the transmembrane and cytosolic domains thereof is used.

[0160] Various assays are optionally used to test the safety of a vaccine. For example, sucrose gradients and neutralization assays are used to test the safety. A sucrose gradient assay is optionally used to determine whether a heterologous protein is inserted in a virion. If the heterologous protein is inserted in the virion, the virion is preferably tested for its ability to cause symptoms in an appropriate animal model since the virus may have acquired new, possibly pathological, properties.

5.4 Adjuvants and Carrier Molecules

[0161] hEbola-associated antigens are administered with one or more adjuvants. In one embodiment, the hEbola-associated antigen is administered together with a mineral salt adjuvants or mineral salt gel adjuvant. Such mineral salt and mineral salt gel adjuvants include, but are not limited to, aluminum hydroxide (ALHYDROGEL, REHYDRAGEL), aluminum phosphate gel, aluminum hydroxyphosphate (ADJU-PHOS), and calcium phosphate.

[0162] In another embodiment, hEbola-associated antigen is administered with an immunostimulatory adjuvant. Such class of adjuvants include, but are not limited to, cytokines (e.g., interleukin-2, interleukin-7, interleukin-12, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ interleukin-1β (IL-1β), and IL-1β peptide or Sclavo Peptide), cytokine-containing liposomes, triterpenoid glycosides or saponins (e.g., QuilA and QS-21, also sold under the trademark STIMULON, ISCOPREP), Muramyl Dipeptide (MDP) derivatives, such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (Threonyl-MDP, sold under the trademark TERMURTIDE), GMDP, N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-s- -n-glycero-3-hydroxy phosphoryloxy)-ethylamine, muramyl tripeptide phosphatidylethanolamine (MTP-PE), unmethylated CpG dinucleotides and oligonucleotides, such as bacterial DNA and fragments thereof, LPS, monophosphoryl Lipid A (3D-MLA sold under the trademark MPL), and polyphosphazenes.

[0163] In another embodiment, the adjuvant used is a particular adjuvant, including, but not limited to, emulsions, e.g., Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, squalene or squalane oil-in-water adjuvant formulations, such as SAF and MF59, e.g., prepared with block-copolymers, such as L-121 (polyoxypropylene/polyoxyetheylene) sold under the trademark PLURONIC L-121, Liposomes, Virosomes, cochleates, and immune stimulating complex, which is sold under the trademark ISCOM.

[0164] In another embodiment, a microparticular adjuvant is used. Microparticular adjuvants include, but are not limited to, biodegradable and biocompatible polyesters, homo- and copolymers of lactic acid (PLA) and glycolic acid (PGA), poly(lactide-co-glycolides) (PLGA) microparticles, polymers that self-associate into particulates (poloxamer particles), soluble polymers (polyphosphazenes), and virus-like particles (VLPs) such as recombinant protein particulates, e.g., hepatitis B surface antigen (HbsAg).

[0165] Yet another class of adjuvants that are optionally used include mucosal adjuvants, including but not limited to heat-labile enterotoxin from Escherichia coli (LT), cholera holotoxin (CT) and cholera Toxin B Subunit (CTB) from Vibrio cholerae, mutant toxins (e.g., LTK63 and LTR72), microparticles, and polymerized liposomes.

[0166] In other embodiments, any of the above classes of adjuvants are optionally used in combination with each other or with other adjuvants. For example, non-limiting examples of combination adjuvant preparations used to administer the hEbola-associated antigens of the invention include liposomes containing immunostimulatory protein, cytokines, T-cell and/or B-cell peptides, or microbes with or without entrapped IL-2 or microparticles containing enterotoxin. Other adjuvants known in the art are also included within the scope of the invention (see Vaccine Design: The Subunit and Adjuvant Approach, Chap. 7, Michael F. Powell and Mark J. Newman (eds.), Plenum Press, New York, 1995, which is incorporated herein in its entirety).

[0167] The effectiveness of an adjuvant is illustratively determined by measuring the induction of antibodies directed against an immunogenic polypeptide containing a hEbola polypeptide epitope, the antibodies resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants.

[0168] The polypeptides are optionally formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid additional salts (formed with free amino groups of the peptide) and which are formed with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with free carboxyl groups are optionally derived from inorganic bases, such as, for example, sodium potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[0169] The vaccines of the invention are preferably multivalent or univalent. Multivalent vaccines are made from recombinant viruses that direct the expression of more than one antigen.

[0170] Many methods are operable herein to introduce the vaccine formulations of the invention; these include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal routes, and via scarification (scratching through the top layers of skin, e.g., using a bifurcated needle).

[0171] The patient to which the vaccine is administered is preferably a mammal, most preferably a human, but is also optionally a non-human animal including but not limited to lower primates, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice and rats.

Preparation of Antibodies

[0172] Antibodies that specifically recognize a polypeptide of the invention, such as, but not limited to, polypeptides including the sequence of SEQ ID NOs: 2-9, 59, or 11-19 and other polypeptides as described herein, or hEbola epitope or antigen-binding fragments thereof are used in a preferred embodiment for detecting, screening, and isolating the polypeptide of the invention or fragments thereof, or similar sequences that might encode similar enzymes from the other organisms. For example, in one specific embodiment, an antibody which immunospecifically binds hEbola epitope, or a fragment thereof, is used for various in vitro detection assays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, western blot, etc., for the detection of a polypeptide of the invention or, preferably, hEbola, in samples, for example, a biological material, including cells, cell culture media (e.g., bacterial cell culture media, mammalian cell culture media, insect cell culture media, yeast cell culture media, etc.), blood, plasma, serum, tissues, sputum, naseopharyngeal aspirates, etc.

[0173] Antibodies specific for a polypeptide of the invention or any epitope of hEbola are optionally generated by any suitable method known in the art. Polyclonal antibodies to an antigen of interest, for example, the hEbola virus from Deposit Accession No. 200706291, or including a nucleotide sequence of SEQ ID NOs: 1 or 10, are optionally produced by various procedures well known in the art. For example, an antigen is optionally administered to various host animals including, but not limited to, rabbits, mice, rats, etc., to induce the production of antisera containing polyclonal antibodies specific for the antigen. Various adjuvants are optionally used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful adjuvants for humans such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0174] Monoclonal antibodies are optionally prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. In one example, monoclonal antibodies are produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which are incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

[0175] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice are immunized with an antigen of interest or a cell expressing such an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, is optionally generated by inoculating mice intraperitoneally with positive hybridoma clones.

[0176] Antibody fragments which recognize specific epitopes are optionally generated by known techniques. For example, Fab and F(ab')₂ fragments are illustratively produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments). F(ab')₂ fragments preferably contain the complete light chain, and the variable region, the CH1 region and the hinge region of the heavy chain.

[0177] The antibodies of the invention or fragments thereof are optionally produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

[0178] The nucleotide sequence encoding an antibody is obtained from any information available to those skilled in the art (i.e., from Genbank, the literature, or by routine cloning and sequence analysis). If a clone containing a nucleic acid encoding a particular antibody or an epitope-binding fragment thereof is not available, but the sequence of the antibody molecule or epitope-binding fragment thereof is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR are optionally then cloned into replicable cloning vectors using any method known in the art.

[0179] Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody is optionally manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., supra; and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence by, for example, introducing amino acid substitutions, deletions, and/or insertions into the epitope-binding domain regions of the antibodies or any portion of antibodies which may enhance or reduce biological activities of the antibodies.

[0180] Recombinant expression of an antibody requires construction of an expression vector containing a nucleotide sequence that encodes the antibody. Once a nucleotide sequence encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof has been obtained, the vector for the production of the antibody molecule is optionally produced by recombinant DNA technology using techniques known in the art as discussed in the previous sections. Methods which are known to those skilled in the art are optionally used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The nucleotide sequence encoding the heavy-chain variable region, light-chain variable region, both the heavy-chain and light-chain variable regions, an epitope-binding fragment of the heavy- and/or light-chain variable region, or one or more complementarity determining regions (CDRs) of an antibody are optionally cloned into such a vector for expression. Thus, prepared expression vector is optionally then introduced into appropriate host cells for the expression of the antibody. Accordingly, the invention includes host cells containing a polynucleotide encoding an antibody specific for the polypeptides of the invention or fragments thereof.

[0181] The host cell is optionally co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors illustratively contain identical selectable markers which enable equal expression of heavy and light chain polypeptides or different selectable markers to ensure maintenance of both plasmids. Alternatively, a single vector is optionally used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature, 322:52, 1986; and Kohler, Proc. Natl. Acad. Sci. USA, 77:2 197, 1980). The coding sequences for the heavy and light chains optionally include cDNA or genomic DNA.

[0182] In another embodiment, antibodies are generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage is utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest is optionally selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage, including fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods, 182:41-50, 1995; Ames et al., J. Immunol. Methods, 184:177-186, 1995; Kettleborough et al., Eur. J. Immunol., 24:952-958, 1994; Persic et al., Gene, 187:9-18, 1997; Burton et al., Advances in Immunology, 57:191-280, 1994; PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0183] As described in the above references, after phage selection, the antibody coding regions from the phage is optionally isolated and used to generate whole antibodies, including human antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab' and F(ab')₂ fragments are optionally employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869, 1992; and Sawai et al., AJR1, 34:26-34, 1995; and Better et al., Science, 240:1041-1043, 1988 (each of which is incorporated by reference in its entirety). Examples of techniques operable to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88, 1991; Shu et al., PNAS, 90:7995-7999, 1993; and Skerra et al., Science, 240:1038-1040, 1988.

[0184] Once an antibody molecule of the invention has been produced by any methods described above, or otherwise known in the art, it is then optionally purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A or Protein G purification, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique(s) for the purification of proteins. Further, the antibodies of the present invention or fragments thereof are optionally fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification. Illustrative examples include 6×His tag, FLAG tag, biotin, avidin, or other system.

[0185] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it is preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a constant region derived from a human immunoglobulin. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science, 229:1202, 1985; Oi et al., BioTechniques, 4:214 1986; Gillies et al., J. Immunol. Methods, 125:191-202, 1989; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody molecules from non-human species that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature, 332:323, 1988, which are incorporated herein by reference in their entireties. Antibodies are humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology, 28(4/5):489-498, 1991; Studnicka et al., Protein Engineering, 7(6):805-814, 1994; Roguska et al., Proc Natl. Acad. Sci. USA, 91:969-973, 1994), and chain shuffling (U.S. Pat. No. 5,565,332), all of which are hereby incorporated by reference in their entireties.

[0186] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies are made by a variety of methods known in the art illustratively including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741, each of which is incorporated herein by reference in its entirety.

[0187] Human antibodies are also illustratively produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol., 13:65-93, 1995. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference herein in their entireties. In addition, companies such as Abgenix, Inc. (Fremont, Calif.), Medarex (NJ) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[0188] Completely human antibodies which recognize a selected epitope are optionally generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology, 12:899-903, 1988).

[0189] Antibodies fused or conjugated to heterologous polypeptides are optionally used in in vitro immunoassays and in purification methods (e.g., affinity chromatography) known in the art. See e.g., PCT publication No. WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett., 39:91-99, 1994; U.S. Pat. No. 5,474,981; Gillies et al., PNAS, 89:1428-1432, 1992; and Fell et al., J. Immunol., 146:2446-2452, 1991, which are incorporated herein by reference in their entireties.

[0190] Antibodies may also be illustratively attached to solid supports, which are particularly useful for immunoassays or purification of the polypeptides of the invention or fragments, derivatives, analogs, or variants thereof, or similar molecules having the similar enzymatic activities as the polypeptide of the invention. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Pharmaceutical Compositions and Kits

[0191] The present invention encompasses pharmaceutical compositions including antiviral agents of the present invention. In a specific embodiment, the antiviral agent is preferably an antibody which immunospecifically binds and neutralizes the hEbola virus or variants thereof, or any proteins derived therefrom. In another specific embodiment, the antiviral agent is a polypeptide or nucleic acid molecule of the invention. The pharmaceutical compositions have utility as an antiviral prophylactic agent are illustratively administered to a subject where the subject has been exposed or is expected to be exposed to a virus.

[0192] Various delivery systems are known and operable to administer the pharmaceutical composition of the invention, illustratively, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, and receptor mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429 4432). Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and optionally administered together with other biologically active agents. Administration is systemic or local. In a preferred embodiment, it is desirable to introduce the pharmaceutical compositions of the invention into the lungs by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0193] In a specific embodiment, it is desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This administration may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, by means of nasal spray, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) infected tissues.

[0194] In another embodiment, the pharmaceutical composition is delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0195] In yet another embodiment, the pharmaceutical composition is delivered in a controlled release system. In one embodiment, a pump is used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials are used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system is placed in proximity of the composition's target, i.e., the lung, thus, requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[0196] Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)) the contents of which are incorporated herein by reference.

[0197] The pharmaceutical compositions of the present invention illustratively include a therapeutically effective amount of a live attenuated, inactivated or killed West African hEbola virus, or recombinant or chimeric hEbola virus, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical carriers are illustratively sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are optionally employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, also contains wetting or emulsifying agents, or pH buffering agents. These compositions optionally take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like. The composition is optionally formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation illustratively includes standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The formulation should suit the mode of administration.

[0198] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The composition also includes an optional solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline is optionally provided so that the ingredients may be mixed prior to administration.

[0199] The pharmaceutical compositions of the invention are illustratively formulated as neutral or salt forms. Pharmaceutically acceptable salts illustratively include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2 ethylamino ethanol, histidine, procaine, etc.

[0200] The amount of the pharmaceutical composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays are optionally employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20 to 500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems.

[0201] Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

[0202] The invention also provides a pharmaceutical pack or kit including one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) is a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In a preferred embodiment, the kit contains an antiviral agent of the invention, e.g., an antibody specific for the polypeptides encoded by a nucleotide sequence of SEQ ID NOs: 1 or 10, or as shown in SEQ ID NOs: 2-9, 59, or 11-19, or any hEbola epitope, or a polypeptide or protein of the present invention, or a nucleic acid molecule of the invention, alone or in combination with adjuvants, antivirals, antibiotics, analgesic, bronchodilators, or other pharmaceutically acceptable excipients.

[0203] The present invention further encompasses kits including a container containing a pharmaceutical composition of the present invention and instructions for use.

Detection Assays

[0204] The present invention provides a method for detecting an antibody, which immunospecifically binds to the hEbola virus, in a biological sample, including for example blood, serum, plasma, saliva, urine, feces, etc., from a patient suffering from hEbola infection, and/or hemorrhagic fever. In a specific embodiment, the method including contacting the sample with the hEbola virus, for example, of Deposit Accession No. 200706291, or having a genomic nucleic acid sequence of SEQ ID NOs: 1 or 10, directly immobilized on a substrate and detecting the virus-bound antibody directly or indirectly by a labeled heterologous anti-isotype antibody. In another specific embodiment, the sample is contacted with a host cell which is infected by the hEbola virus, for example, of Deposit Accession No. 200706291, or having a genomic nucleic acid sequence of SEQ ID NOs: 1 or 10, and the bound antibody is optionally detected by immunofluorescent assay.

[0205] An exemplary method for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample involves obtaining a biological sample from various sources and contacting the sample with a compound or an agent capable of detecting an epitope or nucleic acid (e.g., mRNA, genomic DNA) of the hEbola virus such that the presence of the hEbola virus is detected in the sample. A preferred agent for detecting hEbola mRNA or genomic RNA of the invention is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic RNA encoding a polypeptide of the invention. The nucleic acid probe is, for example, a nucleic acid molecule including the nucleotide sequence of SEQ ID NOs: 1 or 10, a complement thereof, or a portion thereof, such as an oligonucleotide of at least 15, 20, 25, 30, 50, 100, 250, 500, 750, 1000 or more contiguous nucleotides in length and sufficient to specifically hybridize under stringent conditions to a hEbola mRNA or genomic RNA.

[0206] As used herein, the term "stringent conditions" describes conditions for hybridization and washing under which nucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to each other typically remain hybridized to each other. Such hybridization conditions are described in, for example but not limited to, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6; Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986), pp. 75 78, and 84 87; and Molecular Cloning, Cold Spring Harbor Laboratory, N.Y. (1982), pp. 387 389, and are well known to those skilled in the art. A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC), 0.5% SDS at about 68° C. followed by one or more washes in 2×SSC, 0.5% SDS at room temperature. Another preferred, non-limiting example of stringent hybridization conditions is hybridization in 6×SSC at about 45° C. followed by one or more washes in 0.2×SSC, 0.1% SDS at 50 to 65° C.

[0207] A nucleic acid probe, polynucleotide, oligonucleotide, or other nucleic acid is preferably purified. An "isolated" or "purified" nucleotide sequence is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the nucleotide is derived, or is substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a nucleotide/oligonucleotide in which the nucleotide/oligonucleotide is separated from cellular components of the cells from which it is isolated or produced. Thus, a nucleotide/oligonucleotide that is substantially free of cellular material includes preparations of the nucleotide having less than about 30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of contaminating material. When nucleotide/oligonucleotide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly, such preparations of the nucleotide/oligonucleotide have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the nucleotide/oligonucleotide of interest. In a preferred embodiment of the present invention, the nucleotide/oligonucleotide is isolated or purified.

[0208] In another preferred specific embodiment, the presence of hEbola virus is detected in the sample by a reverse transcription polymerase chain reaction (RT-PCR) using the primers that are constructed based on a partial nucleotide sequence of the genome of hEbola virus, for example, that of Deposit Accession No. 200706291, or having a genomic nucleic acid sequence of SEQ ID NOs: 1 or 10. In a non-limiting specific embodiment, preferred primers to be used in a RT-PCR method are the primers are described in detail herein.

[0209] In more preferred specific embodiment, the present invention provides a real-time quantitative PCR assay to detect the presence of hEbola virus in a biological sample by subjecting the cDNA obtained by reverse transcription of the extracted total RNA from the sample to PCR reactions using the specific primers described in detail herein, and a fluorescence dye, such as SYBR® Green I, which fluoresces when bound nonspecifically to double-stranded DNA. The fluorescence signals from these reactions are captured at the end of extension steps as PCR product is generated over a range of the thermal cycles, thereby allowing the quantitative determination of the viral load in the sample based on an amplification plot.

[0210] A preferred agent for detecting hEbola is an antibody that specifically binds a polypeptide of the invention or any hEbola epitope, preferably an antibody with a detectable label. Antibodies are illustratively polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) is operable herein.

[0211] The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, optionally via a linker, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it is detectable with fluorescently labeled streptavidin. The detection method of the invention is optionally used to detect mRNA, protein (or any epitope), or genomic RNA in a sample in vitro as well as in vivo. Exemplary in vitro techniques for detection of mRNA include northern hybridizations, in situ hybridizations, RT-PCR, and RNase protection. In vitro techniques for detection of an epitope of hEbola illustratively include enzyme linked immunosorbent assays (ELISAs), western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of genomic RNA include northern hybridizations, RT-PCT, and RNase protection. Furthermore, in vivo techniques for detection of hEbola include introducing into a subject organism a labeled antibody directed against the polypeptide. In one embodiment, the antibody is labeled with a radioactive marker whose presence and location in the subject organism is detected by standard imaging techniques, including autoradiography.

[0212] In a specific embodiment, the methods further involve obtaining a control sample from a control subject, contacting the control sample with a compound or agent capable of detecting hEbola, e.g., a polypeptide of the invention or mRNA or genomic RNA encoding a polypeptide of the invention, such that the presence of hEbola or the polypeptide or mRNA or genomic RNA encoding the polypeptide is detected in the sample, and comparing the absence of hEbola or the polypeptide or mRNA or genomic RNA encoding the polypeptide in the control sample with the presence of hEbola, or the polypeptide or mRNA or genomic DNA encoding the polypeptide in the test sample.

[0213] The invention also encompasses kits for detecting the presence of hEbola or a polypeptide or nucleic acid of the invention in a test sample. The kit illustratively includes a labeled compound or agent capable of detecting hEbola or the polypeptide or a nucleic acid molecule encoding the polypeptide in a test sample and, in certain embodiments, a means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits optionally include instructions for use.

[0214] For antibody-based kits, the kit illustratively includes: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide of the invention or hEbola epitope; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is preferably conjugated to a detectable agent.

[0215] For oligonucleotide-based kits, the kit illustratively includes: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a polypeptide of the invention or to a sequence within the hEbola genome; or (2) a pair of primers useful for amplifying a nucleic acid molecule containing an hEbola sequence. The kit optionally includes a buffering agent, a preservative, or a protein stabilizing agent. The kit optionally includes components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit optionally contains a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for use.

Screening Assays to Identify Antiviral Agents

[0216] The invention provides methods for the identification of a compound that inhibits the ability of hEbola virus to infect a host or a host cell. In certain embodiments, the invention provides methods for the identification of a compound that reduces the ability of hEbola virus to replicate in a host or a host cell. Any technique well known to the skilled artisan is illustratively used to screen for a compound useful to abolish or reduce the ability of hEbola virus to infect a host and/or to replicate in a host or a host cell.

[0217] In certain embodiments, the invention provides methods for the identification of a compound that inhibits the ability of hEbola virus to replicate in a mammal or a mammalian cell. More specifically, the invention provides methods for the identification of a compound that inhibits the ability of hEbola virus to infect a mammal or a mammalian cell. In certain embodiments, the invention provides methods for the identification of a compound that inhibits the ability of hEbola virus to replicate in a mammalian cell. In a specific embodiment, the mammalian cell is a human cell.

[0218] In another embodiment, a cell is contacted with a test compound and infected with the hEbola virus. In certain embodiments, a control culture is infected with the hEbola virus in the absence of a test compound. The cell is optionally contacted with a test compound before, concurrently with, or subsequent to the infection with the hEbola virus. In a specific embodiment, the cell is a mammalian cell. In an even more specific embodiment, the cell is a human cell. In certain embodiments, the cell is incubated with the test compound for at least 1 minute, at least 5 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, or at least 1 day. The titer of the virus is optionally measured at any time during the assay. In certain embodiments, a time course of viral growth in the culture is determined. If the viral growth is inhibited or reduced in the presence of the test compound, the test compound is identified as being effective in inhibiting or reducing the growth or infection of the hEbola virus. In a specific embodiment, the compound that inhibits or reduces the growth of the hEbola virus is tested for its ability to inhibit or reduce the growth rate of other viruses to test its specificity for the hEbola virus.

[0219] In one embodiment, a test compound is administered to a model animal and the model animal is infected with the hEbola virus. In certain embodiments, a control model animal is infected with the hEbola virus without the administration of a test compound. The test compound is optionally administered before, concurrently with, or subsequent to the infection with the hEbola virus. In a specific embodiment, the model animal is a mammal. In an even more specific embodiment, the model animal is, but is not limited to, a cotton rat, a mouse, or a monkey. The titer of the virus in the model animal is optionally measured at any time during the assay. In certain embodiments, a time course of viral growth in the culture is determined. If the viral growth is inhibited or reduced in the presence of the test compound, the test compound is identified as being effective in inhibiting or reducing the growth or infection of the hEbola virus. In a specific embodiment, the compound that inhibits or reduces the growth of the hEbola in the model animal is tested for its ability to inhibit or reduce the growth rate of other viruses to test its specificity for the hEbola virus.

[0220] According to the method of the invention, a human or an animal is optionally treated for for EboBun or EboIC, other viral infection or bacterial infection by administering an effective amount of an inventive therapeutic composition. Preferably, a vaccine is administered prophylactically. An "effective amount" is an amount that will induce an immune response in a subject. Illustratively, an effective amount of the compositions of this invention ranges from nanogram/kg to milligram/kg amounts for young children and adults. Equivalent dosages for lighter or heavier body weights can readily be determined. The dose should be adjusted to suit the individual to whom the composition is administered and will vary with age, weight and metabolism of the individual. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular peptide or polypeptide used, its mode of administration and the like. An appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. One skilled in the art will realize that dosages are best optimized by the practicing physician or veterinarian and methods for determining dose amounts and regimens and preparing dosage forms are described, for example, in Remington's Pharmaceutical Sciences, (Martin, E. W., ed., latest edition), Mack Publishing Co., Easton, Pa. Preferably, a single administration is operable to induce an immune response.

[0221] Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.

[0222] Embodiments of inventive compositions and methods are illustrated in the following detailed examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.

EXAMPLES

Example 1

Newly Discovered Ebola Virus Associated with Hemorrhagic Fever Outbreak in Bundibugyo, Uganda

[0223] In late November 2007 HF cases were reported in the townships of Bundibugyo and Kikyo in Bundibugyo District, Western Uganda (FIG. 1A). These samples were assayed as described by Towner, J S, et al., PLoS Pathog, 2008 November; 4(11): e1000212, the contents of which are incorporated herein by reference for methods, results, reagents, and all other aspects of the publication. A total of 29 blood samples were initially collected from suspect cases and showed evidence of acute ebolavirus infection in eight specimens using a broadly reactive ebolavirus antigen capture assay known to cross-react with the different ebolavirus species' and an IgM capture assay based on Zaire ebolavirus reagents (Table 1). These specimens were negative when initially tested with highly sensitive real-time RT-PCR assays specific for all known Zaire and Sudan ebolaviruses and marburgviruses. However, further evidence of acute ebolavirus infection was obtained using a traditionally less sensitive (relative to the real-time RT-PCR assays) but more broadly reactive filovirus L gene-specific RT-PCR assay (1 specimen) (Table 1). Sequence analysis of the PCR fragment (400 bp of the virus L gene) revealed the reason for the initial failure of the real-time RT-PCR assays, as the sequence was distinct from that of the 4 known species of ebolavirus, although distantly related to Cote d'Ivoire ebolavirus. In total, 9 of 29 specimens showed evidence of ebolavirus infection, and all tests were negative for marburgvirus (data not shown).

[0224] Approximately 70% of the virus genome was rapidly sequenced from total RNA extracted from a patient serum (#200706291) using a newly established metagenomics pyrosequencing method (454 Life Sciences) which involves successive rounds of random DNA amplification⁸. Using the newly derived draft sequence, a real-time RT-PCR assay specific for the NP gene of this virus was quickly developed and evaluated. The assay was shown to have excellent sensitivity (Table 1), finding positive all the initial six samples that tested positive by either virus antigen capture (five specimens) or virus isolation assays (four specimens). The antigen-capture, IgM, IgG and newly designed real-time PCR assays were quickly transferred to the Uganda Virus Research Institute during the course of the outbreak to facilitate rapid identification and isolation of Ebola cases in the affected area for efficient control of the outbreak. The outbreak continued through late December 2007, and resulted in 149 suspected cases and 37 deaths⁹.

[0225] Table 1. Ebolavirus diagnostic results of initial 29 specimens obtained from Bundibugyo District with numerical specimen numbers assigned. RT-PCR refers to results obtained from conventional PCR using the broadly reactive Filo A/B primers¹³. Ag, IgM, and IgG refer to results from ELISA-based assays¹⁰, 11 with Zaire ebolavirus reagents while virus isolation refers to culture attempts on Vero E6 cells'². Q-RT-PCR refers to results obtained using the optimized Bundibugyo ebolavirus specific real-time RT-PCR assay with cycle threshold (Ct) values of positive (Pos) samples indicated in the far right column. * Specimen #200706291 is the clinical sample from which prototype isolate #811250 was obtained.

TABLE-US-00001 TABLE 1 Sample RT- Virus Q- RT- No. PCR Ag IgM IgG Isolation PCR Ct 200706288 neg neg neg neg neg neg 40 200706289 neg neg neg neg neg neg 40 200706290 neg neg neg neg neg neg 40 200706291* Pos Pos neg neg Pos Pos 23.64 200706292 neg neg neg neg neg neg 40 200706293 neg neg neg neg neg neg 40 200706294 neg neg neg neg neg neg 40 200706295 neg neg neg neg neg neg 40 200706296 neg neg Pos Pos neg neg 40 200706297 neg neg Pos Pos neg neg 40 200706298 neg Pos Pos Pos neg Pos 34.83 200706299 neg neg Pos Pos neg neg 40 200706300 neg neg neg neg neg neg 40 200706301 neg neg neg neg neg neg 40 200706302 neg Pos Pos neg neg Pos 35.01 200706303 neg neg neg neg neg neg 40 200706304 neg neg neg neg Pos Pos 38.18 200706305 neg neg neg neg neg neg 40 200706306 neg neg neg neg neg neg 40 200706307 neg neg neg neg neg neg 40 200706320 ND Pos neg neg Pos Pos 30.24 200706321 ND neg neg neg neg neg 40 200706322 ND neg neg neg neg neg 40 200706323 ND neg neg neg neg neg 40 200706324 ND neg neg neg neg neg 40 200706325 ND neg neg neg neg neg 40 200706326 ND neg neg neg neg neg 40 200706327 ND Pos neg neg Pos Pos 34.41 200706328 ND neg neg neg neg neg 40

[0226] The entire genome sequence of this virus was completed using a classic primer walking sequencing approach on RNA. The complete genome of the Eb ebolavirus was not available, so it too was derived by a similar combination of random primed pyrosequencing and primer walking approaches. Acquisition of these sequences allowed for the first time the phylogenetic analysis of the complete genomes of representatives of all known species of Ebola and Marburg viruses. The analysis revealed that the newly discovered virus differed from the four existing ebolavirus species (FIG. 1), with approximately 32% nucleotide difference from even the closest relative, EboIC (Table 2). Similar complete genome divergence (35-45%) is seen between the previously characterized ebolavirus species.

[0227] Table 2. Identity matrix based on comparisons of full-length genome sequences of Zaire ebolaviruses 1976 (Genbank accession number NC_--002549) and 1995 (Genbank accession number AY354458), Sudan ebolavirus 2000 (Genbank accession number NC_--006432), Cote d'Ivoire ebolavirus 1994 (SEQ ID NO: 10), Reston ebolavirus 1989 (Genbank accession number NC_--004161), and Bundibugyo ebolavirus 2007 (SEQ ID NO: 1).

TABLE-US-00002 TABLE 2 Zaire Sudan EboIC EboBun Reston '95 '00 '94 '07 '89 Zaire '76 .988 .577 .630 .632 .581 Zaire '95 .577 .631 .633 .581 Sudan '00 .577 .577 .609 EboIC '94 .683 .575 EboBun '07 .576

[0228] The material and information obtained from the discovery of the new unique virus EboBun and the realization that together with EboIC these viruses represent a Glade of Bundibungyo-Ivory Coast Ebola virus species is valuable, and makes possible the development of clinical, diagnostic and research tools directed to human hEbola infection.

Material and Methods

[0229] Ebolavirus Detection and Virus Isolation.

[0230] Several diagnostic techniques were used for each sample: (i) antigen capture, IgG, and IgM assays were performed as previously described¹¹ (ii) virus isolation attempts were performed on Vero E6 cells'² and monitored for 14 days; (iii) RNA was extracted and tested for Zaire¹⁶ and Sudan ebolavirus and marburgvirus⁴ using real-time quantitative RT-PCR assays designed to detect all known species of each respective virus species the primers/probe for the Sudan ebolavirus assay were EboSudBMG 1(+) 5'-GCC ATG GIT TCA GGT TTG AG-3' (SEQ ID NO: 21), EboSudBMG 1(-) 5'-GGT IAC ATT GGG CAA CAA TTC A-3' (SEQ ID NO: 22) and Ebola Sudan BMG Probe 5'FAM-AC GGT GCA CAT TCT CCT TTT CTC GGA-BHQ1 (SEQ ID NO: 23)]; (iv) the conventional RT-PCR was performed with the filo A/B primer set as previously described¹⁶ using Superscript III (Invitrogen) according to the manufacturer's instructions. The specimen 200706291 was selected as the reference sample for further sequence analysis.

[0231] Genome Sequencing.

[0232] Pyrosequencing was carried out utilizing the approach developed by 454 Life Sciences, and the method described by Cox-Foster et al.⁸ Subsequent virus whole genome primer walking was performed as previously described¹⁷ but using the primers specific for Bundibugyo ebolavirus RT-PCR amplification. In total, the entire virus genome was amplified in six overlapping RT-PCR fragments (all primers listed 5' to 3'): fragment A (predicted size 2.7 kb) was amplified using forward-GTGAGACAAAGAATCATTCCTG (SEQ ID NO: 24) with reverse-CATCAATTGCTCAGAGATCCACC (SEQ ID NO: 25); fragment B (predicted size 3.0 kb) was amplified using forward-CCAACAACACTGCATGTAAGT (SEQ ID NO: 26) with reverse-AGGTCGCGTTAATCTTCATC (SEQ ID NO: 27); fragment C (predicted size 3.5 kb) was amplified using forward-GATGGTTGAGTTACTTTCCGG (SEQ ID NO: 28) with reverse-GTCTTGAGTCATCAATGCCC (SEQ ID NO: 29); fragment D (predicted size 3.1 kb) was amplified using forward-CCACCAGCACCAAAGGAC (SEQ ID NO: 30) with reverse-CTATCGGCAATGTAACTATTGG (SEQ ID NO: 31); fragment E (predicted size 3.4 kb) was amplified using forward-GCCGTTGTAGAGGACACAC (SEQ ID NO: 32) with reverse-CACATTAAATTGTTCTAACATGCAAG (SEQ ID NO: 33) and fragment F (predicted size 3.5 kb) was amplified using forward-CCTAGGTTATTTAGAAGGGACTA (SEQ ID NO: 34) with reverse-GGT AGA TGT ATT GAC AGC AAT ATC (SEQ ID NO: 35).

[0233] The exact 5' and 3' ends of Bundibugyo ebolavirus were determined by 3' RACE from virus RNA extracted from virus infected Vero E6 cell monolayers using TriPure isolation reagent. RNAs were then polyadenylated in vitro using A-Plus poly(A) polymerase tailing kit (Epicenter Biotechnologies) following the manufacturer's instructions and then purified using an RNeasy kit (Qiagen) following standard protocols. Ten microliters of in vitro polyadenylated RNA were added as template in RT-PCR reactions, using SuperScript III One-Step RT-PCR system with Platinum Taq High Fidelity (Invitrogen) following the manufacturer's protocol. Two parallel RT-PCR reactions using the oligo(dT)-containing 3'RACE-AP primer (Invitrogen) mixed with 1 of 2 viral specific primers, Ebo-U 692(-) ACAAAAAGCTATCTGCACTAT (SEQ ID NO: 36) and Ebo-V18269(+) CTCAGAAGCAAAATTAATGG (SEQ ID NO: 37), generated ˜700 nt long fragments containing the 3' ends of either genomic and antigenomic RNAs. The resulting RT-PCR products were analyzed by agarose electrophoresis, and DNA bands of the correct sizes were purified using QIAquick Gel Extraction Kit (Qiagen) and sequenced using standard protocols (ABI).

[0234] The nucleotide sequence of the Cote d'Ivoire ebolavirus (EboIC) isolate RNA was initially determined using the exact same pyrosequencing strategy as that used for Bundibugyo ebolavirus described above. This method generated sequence for approximately 70% of the entire genome. This draft sequence was then used to design a whole genome primer walking strategy for filling any gaps and confirming the initial sequence. The following Cote d'Ivoire ebolavirus-specific primers were used to generate RT-PCR fragments, designated A-F, as follows: Fragment A (predicted size 3.0 kb) was amplified using forward-GTGTGCGAATAACTATGAGGAAG (SEQ ID NO: 38) and reverse-GTCTGTGCAATGTTGATGAAGG (SEQ ID NO: 39); Fragment B (predicted size 3.2 kb) was amplified using forward-CATGAAAACCACACTCAACAAC (SEQ ID NO: 40) and reverse-GTTGCCTTAATCTTCATCAAGTTC (SEQ ID NO: 41); Fragment C (predicted size 3.0 kb) was amplified using forward-GGCTATAATGAATTTCCTCCAG (SEQ ID NO: 42) and reverse-CAAGTGTATTTGTGGTCCTAGC (SEQ ID NO: 43); fragment D (predicted size 3.5 kb) was amplified using forward-GCTGGAATAGGAATCACAGG (SEQ ID NO: 44) and reverse-CGGTAGTCTACAGTTCTTTAG (SEQ ID NO: 45); fragment E (predicted size 4.0 kb) was amplified using forward-GACAAAGAGATTAGATTAGCTATAG (SEQ ID NO: 46) and reverse-GTAATGAGAAGGTGTCATTTGG (SEQ ID NO: 47); fragment F (predicted size 2.9 kb) was amplified using forward-CACGACTTAGTTGGACAATTGG (SEQ ID NO: 48) and reverse-CAGACACTAATTAGATCTGGAAG (SEQ ID NO: 49); fragment G (predicted size 1.3 kb) was amplified using forward-CGGACACACAAAAAGAAWRAA (SEQ ID NO: 50) and reverse-CGTTCTTGACCTTAGCAGTTC (SEQ ID NO: 51); and fragment H (predicted size 2.5 kb) was amplified using forward-GCACTATAAGCTCGATGAAGTC (SEQ ID NO: 52) and reverse-TGGACACACAAAAARGARAA (SEQ ID NO: 53). A gap in the sequence contig was located between fragments C and D and this was resolved using the following primers to generate a predicted fragment of 1.5 kb: forward-CTGAGAGGATCCAGAAGAAAG (SEQ ID NO: 54) and reverse-GTGTAAGCGTTGATATACCTCC (SEQ ID NO: 55). The terminal ˜20 nucleotides of the sequence were not experimentally determined but were inferred by comparing with the other known Ebola genome sequences.

[0235] Bundibugyo ebolavirus Real-Time RT-PCR Assay.

[0236] The primers and probe used in the Bundibugyo ebolavirus specific Q-RT-PCR assay were as follows: EboU965(+): 5'-GAGAAAAGGCCTGTCTGGAGAA-3' (SEQ ID NO: 56), EboU1039(-): 5'-TCGGGTATTGAATCAGACCTTGTT-3' (SEQ ID NO: 57) and EboU989 Prb: 5'Fam-TTCAACGACAAATCCAAGTGCACGCA-3'BHQ1 (SEQ ID NO 58). Q-RT-PCR reactions were set up using Superscript III One-Step Q-RT-PCR (Invitrogen) according to the manufacturer's instructions and run for 40 cycles with a 58° C. annealing temperature.

[0237] Phylogenetic Analysis.

[0238] Modeltest 3.7¹⁸ was used to examine 56 models of nucleotide substitution to determine the model most appropriate for the data. The General Time Reversible model incorporating invariant sites and a gamma distribution (GTR+I+G) was selected using the Akaike Information Criterion (AIC). Nucleotide frequencies were A=0.3278, C=0.2101, G=0.1832, T=0.2789, the proportion of invariant sites=0.1412, and the gamma shape parameter=1.0593. A maximum likelihood analysis was subsequently performed in PAUP*4.0b10¹⁹ using the GTR+I+G model parameters. Bootstrap support values were used to assess topological support and were calculated based on 1,000 pseudoreplicates²⁰.

[0239] In addition, a Bayesian phylogenetic analysis was conducted in MrBayes 3.2²¹ using the GTR+I+G model of nucleotide substitution. Two simultaneous analyses, each with four Markov chains, were run for 5,000,000 generations sampling every 100 generations. Prior to termination of the run, the AWTY module was used to assess Markov Chain Monte Carlo convergence to ensure that the length of the analysis was sufficient²². Trees generated before the stabilization of the likelihood scores were discarded (burn in =40), and the remaining trees were used to construct a consensus tree. Nodal support was assessed by posterior probability values (>95=statistical support).

Example 2

Immunization against EboBun

[0240] To determine the capability of immunogens to elict an immune response in non-human primates (NHP), 12 cynomolgus macaques, of which 10 are immunized with VSVΔG/EboBunGP either orally (OR; n=4), intranasally (IN; n=4) or intramuscularly (IM; n=2) in accordance with all animal control and safety guidelines and essentially as described by Qiu, X, et al., PLoS ONE. 2009; 4(5): e5547. The remaining 2 control animals are vaccinated intramuscularly with VSVΔG/MARVGP. VSVΔG/MARVGP does not provide heterologous protection against EboBun, therefore these NHPs succumb to EboBun infection. Animals are acclimatized for 14 days prior to infection. Animals are fed and monitored twice daily (pre- and post-infection) and fed commercial monkey chow, treats and fruit. Husbandry enrichment consists of commercial toys and visual stimulation.

[0241] The recombinant VSVΔG/EboBun vaccines are synthesized expressing the EboBun glycoprotein (GP) (SEQ ID NO: 9), soluble glycoprotein (sGP) (SEQ ID NO: 4), or nucleoprotein (NP) (SEQ ID NO: 3). Control VSVΔG/MARVGP vaccines represent the analogous proteins from Lake victoria marburgvirus (MARV) (strain Musoke). The following results for GP are similar for sGP and NP. Vaccines are generated using VSV (Indiana serotype) as described previously. Garbutt, M, et al., J Virol, 2004; 78(10):5458-5465; Schnell, M J, et al., PNAS USA, 1996; 93(21):11359-11365. EboBun challenge virus is passaged in Vero E6 cells prior to challenge, as described previously Jones, S M, et al., Nat Med, 2005; 11(7):786-790; Jahrling, P B, et al., J Infect Dis, 1999; 179 (Suppl 1):S224-34. An EboBun immunogen peptide pool consisting of 15mers with 11 amino acid overlaps (Sigma-Genosys) spanning the entire sequence of the EboBun immunogens and strain Mayinga 1976 GP are used.

[0242] Twelve filovirus naive cynomolgus monkeys randomized into four groups receive 2 ml of 1×10⁷ PFU/ml of vaccine in Dulbecco's modified Eagle's medium (DMEM). Animals in the three experimental groups are vaccinated with either: 1) 2 ml orally (OR) (n=4); 2) 1 ml dripped into each nostril, intranasally (IN) (n=4); or 3) 1 ml each into two sites intramuscularly (IM) (n=2). The two controls are injected intramuscularly with 2 ml of 1×10⁷ PFU/ml of VSVΔG/MARVGP. All animals are challenged intramuscularly 28 days later with 1,000 PFU of EboBun.

[0243] Routine examination is conducted on 0, 2, 4, 6, 10, 14 and 21 days post-vaccination, then 0, 3, 6, 10, 14, 19, 26 days, 6 and 9 months after the EboBun challenge. For the examinations animals are anaesthetized by intramuscular injection with 10 mg/kg of ketaset (Ayerst). Examinations include haematological analysis, monitoring temperature (rectal), respiration rate, lymph nodes, weight, hydration, discharges and mucous membranes. Also, swabs (throat, oral, nasal, rectal, vaginal) and blood samples are collected (4 ml from femoral vein, 1 ml in EDTA vacutainer tube; 3 ml in serum separator vacutainer tube). Cynomolgus monkey PBMCs are isolated using BD CPT sodium citrate Vacutainers (Becton Dickinson) as per manufacturer's protocol.

[0244] All VSVΔG/EboBunGP immunized animals are protected from high dose challenge. These animals show no evidence of clinical illness after vaccination or EboBun challenge. Both control animals demonstrate typical symptoms associated with EboBun HF including fever, macular rashes, lethargy, and unresponsiveness. Continued infection requires euthanization. Hematology analyses at each examination date demonstrate increases in the platelet-crit in the OR and IN groups post-challenge, however, no significant changes are observed in any NHPs post-immunization or in the VSVΔG/EboBunGP immunized NHPs post-challenge.

[0245] EboBun antibody production from humoral antibody response to vaccination and challenge is examined by a virus like particle (VLP) based ELISA assay. Generation of EboBun VLPs is performed by the protocol for ZEBOV as described by Wahl-Jensen, V., et al., J Virol, 2005; 79(4):2413-2419. ELISA is performed by the protocol described by Qiu, X, et al., PLoS ONE. 2009; 4(5): e5547.

[0246] The VSVΔG/MARVGP immunized animals do not develop a detectable antibody response to EboBun. In contrast, potent antibody responses are detected in all VSVΔG/EboBunGP immunized animals independent of immunization route. Between days 14 and 21 post-vaccination, all VSVΔG/EboBunGP immunized NHPs develop high levels of IgA, IgM, and IgG against EboBunGP. After challenge the IgM titres do not exceed the post-vaccination levels, however, IgG and IgA antibody titres are increased peaking 14 days post-challenge then slowly decreasing before maintaining a relatively high antibody titre up to 9 months.

[0247] The level of neutralization antibodies is detected by a EboBun-GFP flow cytometric neutralization assay in serum collected at days 0 and 21 post-vaccination. Samples are assayed in duplicate for their ability to neutralize an infection with EboBun-GFP in VeroE6 cells. Serially diluted serum samples are incubated with an equal volume of EboBun-GFP in DMEM, at 37° C., 5% CO₂ for 1 hr followed by addition of 150 μl per well of a confluent 12 well plate of VeroE6 cells (MOI=0.0005). After 2 hours at 37° C., 5% CO₂, 1 ml of DMEM, 2% fetal bovine serym (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin is added per well and incubated for 5 days. Cells are harvested by removing the culture supernatant, washing with 1 ml PBS, 0.04% EDTA, then adding 800 μl of PBS 0.04% EDTA for 5 minutes at 37° C. before adding 8 ml PBS, 4% paraformaldehyde (PFA) and overnight incubation. The cells are acquired (10,000 events) and analyzed with CellQuest Pro v3.3 on a Becton Dickinson FACSCalibur flow cytometer.

[0248] The OR and IN routes produce EboBunGP-specific neutralizing antibodies with the OR route producing the highest titres post-vaccination. The IM immunization produces detectable levels of neutralizing antibody. In comparison, 3/4 NHPs in the OR group demonstrate a 50% reduction in EboBun-GFP positive cells at a titre of 1:40. Similarly, the IN route results in a reduction of EboBun-GFP positive cells at the 1:40 dilution.

[0249] EboBunGP-specific effector cellular immune responses are determined using IL-2 and IFN-γ ELISPOT assays as described by Qin, X, et al., PLoS ONE. 2009; 4(5): e5547 to determine the number of IL-2 and IFN-γ secreting lymphocytes. Prior to challenge on days 10 to 14 post-vaccination there is a detectable EboBun immunogen-specific IFN-γ response in all immunized animals. The IM route is the most potent, inducing approximately 2-fold more IFN-γ secreting cells than OR (p<0.001) or IN (p=0.043) routes. A strong post-challenge secondary IFN-γ response is induced in all VSVΔG/EboBun immunized animals with the IM route producing the most IFN-γ cells at day 6. By day 10 the OR group demonstrates a stronger response. The IFN-γ in the IN group rises steadily, peaking at day 26 post-challenge with 4.3 and 2 fold more EboBun specific IFN-γ secreting cells than the IM (p=0.003) and OR (p=0.075) group, respectively. All three routes produce strong EboBun-specific IFN-γ responses.

[0250] Post-vaccination, the IM group also has more EboBunGP-specific IL-2 secreting cells than either of the mucosally immunized groups. Post-challenge, the IM route continues to dominate early after challenge peaking on day 10. This difference shows a trend when compared to the IN group (p=0.067) and is significant when compared to the OR group (p<0.001). Additionally, the IN group has more IL-2 producing cells than the OR group (p=0.090) on day 10 post-challenge. By day 26 post-challenge all three routes continue to produce a EboBunGP-specific IL-2 response, however, the IN group response is strongest. At day 26 post-challenge the IN group has the most potent IFN-γ and IL-2 responses, as well as the highest IgA and IgG antibody titre, indicating this immunization route, followed by a EboBun challenge, results in the development of potent and sustained effector responses.

[0251] Absolute lymphocyte numbers for CD3.sup.+, CD4.sup.+, and CD8.sup.+ (CD3.sup.+4.sup.-) T cell populations are determined by flow cytometry. No decrease is observed in the lymphocyte populations for any of the VSVΔG/EboBunGP vaccinated NHPs. In contrast, control animals who are not protected from EboBun show lymphocyte numbers decreased by 28-57%.

[0252] Macrophage numbers are slightly increased in control animals. However, the number of CD14.sup.+ cells is greater in the VSVΔG/EboBunGP vaccinated groups with the IM route showing the most significant increases.

[0253] In order to determine the long term immune response after challenge, EboBunGP-specific CD4.sup.+ and CD8.sup.+ memory T-lymphocytes are examined for their ability to proliferate (CFSE.sup.-) or produce IFN-γ in response to EboBunGP peptides at 6 months post-vaccination. EboBunGP-specific memory responses are observed as a result of vaccination followed by a ZEBOV challenge. These responses persist for at least 6 months. The memory populations in OR and IN inoculation routes demonstrate the greatest potential for proliferation and IFN-γ production post-challenge.

[0254] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.

[0255] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims. All numerical ranges are inclusive of the whole integers and decimals between the endpoints, and inclusive of the endpoints.

REFERENCES

[0256] 1. Suzuki, Y., and Gojobori, T., (1997) The origin and evolution of Ebola and Marburg viruses. Mol Bio Evol, 14(8): 800-806. [0257] 2. Sanchez, A., Geisbert, T. W., Feldmann, H. in Fields Virology (ed. Knipe, D. M., Howley, P. M.) 1409-1448 (Lippincott Williams and Wilkins, Philadelphia, 2007). [0258] 3. Leroy, E. M. et al., (2005) Fruit bats as reservoirs of Ebola virus. Nature, 438, 575-6. [0259] 4. Towner, J. S. et al., (2007) Marburg virus infection detected in a common African bat. PLoS ONE, 2(8), e764. [0260] 5. Swanepoel, R. et al., (2007) Studies of reservoir hosts for Marburg virus. Emerg Infect Dis, 13(12), 1847-51. [0261] 6. Le Guenno, B. et al., (1995) Isolation and partial characterization of a new species of Ebola virus. Lancet, 345(8960), 1271-4. [0262] 7. Ksiazek, T. G. et al. (1999) Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, IgG and IgM antibody findings among EHF patients in Kikwit, 1995. J. Infect Dis 179 (suppl 1), S177-S187. [0263] 8. Cox-Foster, D. L. et al. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318, 283-7. [0264] 9. World Health Organization (2008) Ebola outbreak contained in Uganda. Features, 22 February, www.who.int/features/2008/ebola_outbreak/en/. [0265] 10. Sullivan, N. J., Sanchez, A., Rollin, P. E., Yang, Z.-Y. & Nabel, G. J. (2000) Development of a preventive vaccine for Ebola virus infection in primates. Nature 408, 605-609. [0266] 11. Ksiazek, T. G., West, C. P., Rollin, P. E., Jahrling, P. B. & Peters, C. J. (1999) ELISA for the detection of antibodies to Ebola viruses. J. Infect Dis 179 (suppl 1), S191-S198. [0267] 12. Rodriguez, L. et al. (1999) Persistence and genetic stability of Ebola virus during the outbreak in Kikwit, Zaire 1995. J. Infect Dis 179 (suppl 1), S170-S176. [0268] 13. Sanchez, A. et al. Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates. J. Infect Dis 179 (suppl 1), S164-S169 (1999). [0269] 14. Jones, S. M. et al. (2005) Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat Med 11, 786-90. [0270] 15. Geisbert, T. W. et al. (2008) Recombinant vesicular stomatitis virus vector mediates postexposure protection against Sudan Ebola hemorrhagic fever in nonhuman primates. J Virol 82, 5664-8. [0271] 16. Towner, J. S., Sealy, T. K., Ksiazek, T. & Nichol, S. T. (2007) High-throughput molecular detection of hemorrhagic fever virus threats with applications for outbreak settings. J. Inf Dis 196 (suppl 2), S205-212. [0272] 17. Towner, J. S. et al. (2006) Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. J Virol 80, 6497-516. [0273] 18. Posada, D. & Crandall, K. A. (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817-818. [0274] 19. Swofford, D. L. (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods) version 4.0b10. Sinauer Assoc., Sunderland, Mass. [0275] 20. Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783-791. [0276] 21. Ronquist, F. & Huelsenbeck, J. P. (2003) MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572-1574. [0277] 22. Nylander, J. A. A., Wilgenbusch, J. C., Warren, D. L. & Swofford, D. L. (2008) AWTY (are we there yet?): a system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics 24, 581-583.

Sequence CWU 1

59118940DNABundibugyo ebolavirusmisc_featureFull viral sequence 1cggacacaca aaaagaatga aggattttga atctttattg tgtgcgagta actacgagga 60agattaaaga ttttcctctc attgaaattg aaattgagat tctaatctcg acggatcgat 120ccccaatacc aacactgaga attggcctga agaagtcatc tgctccttgg caaaaccaag 180agcaggccca aagggccatt aggccacatc tgctgagcct gcagaacacg caggacttac 240ttagcagaag agagcgcgtg ccgaaaccag ccaacaaatt gacacagctg ctcactctga 300ccctgaattc ataaacaata ttaagttgac aacagagata ctaatccaat atttggatca 360agaatcaaaa tagtgaaacg actgactatc cctccttaga attagcaaag atccttttgt 420agactattgt gctacattct ctatccaaga cctcaaaatg gatcctcgtc caatcagaac 480ctggatgatg cataacacat ctgaagttga agcagactac cataagattc taactgccgg 540attgtccgtc cagcaaggca ttgtgagaca aagaatcatt cctgtttacc aaatctcaaa 600cctggaggaa gtatgtcaac tcatcataca ggcattcgag gctggcgtcg acttccagga 660tagtgcagat agctttttgt taatgctatg tctgcatcat gcctatcaag gggattataa 720acaatttttg gaaagtaatg cggtaaaata ccttgaaggt catggattcc gttttgagat 780gaagaaaaag gaaggtgtca agcgcctgga ggaactactc cctgctgcct cgagtggaaa 840gaacatcaag agaacattgg ctgcaatgcc cgaggaggaa acaacagaag caaatgctgg 900acaatttctt tcatttgcta gtctgtttct cccaaaattg gttgtcggag aaaaggcctg 960tctggagaag gttcaacgac aaatccaagt gcacgcagaa caaggtctga ttcaataccc 1020gacatcttgg caatcggtgg gacatatgat ggtcatcttc agactaatgc gaaccaactt 1080cctgattaag ttcctcctaa tacatcaagg aatgcatatg gttgcagggc atgatgctaa 1140tgatgccgtc attgccaact ctgtagctca agctcgtttc tccggattgt tgatagtcaa 1200aacagtgctt gatcatatcc tccaaaaaac agagcacgga gttcgcctgc atcccttggc 1260gcgaacagcc aaagtcaaaa atgaggtgag ctcttttaag gccgctttag cctcactagc 1320acaacatgga gaatatgccc cgtttgctcg tctgctgaat ctatctgggg ttaataatct 1380tgagcatggg cttttccctc aactttctgc aattgctttg ggagtagcaa ctgcacatgg 1440gagcactctg gctggagtca atgtaggaga gcaataccaa caactgcgag aagcagccac 1500tgaggccgaa aagcagttgc agaaatatgc tgaatctcgt gaacttgatc acctaggtct 1560tgatgatcag gaaaagaaaa tcctaaaaga cttccatcag aaaaagaatg agatcagctt 1620ccagcagacg acagccatgg tcacactgcg gaaagagaga ttggccaaat tgaccgaagc 1680tattacttcc acctctatcc tcaaaacagg aaggcggtat gatgatgaca atgacatacc 1740ctttccaggg ccaatcaatg ataacgagaa ctctggtcag aacgatgacg atccaacaga 1800ctcccaggat accacaatcc cggatgtaat aatcgatcca aacgatggtg ggtataataa 1860ttacagcgat tatgcaaatg atgctgcaag tgctcctgat gacctagttc tttttgacct 1920tgaggacgag gatgatgctg ataacccggc tcaaaacacg ccagaaaaaa atgatagacc 1980agcaacaaca aagctgagaa atggacagga ccaggatgga aaccaaggcg aaactgcatc 2040cccacgggta gcccccaacc aatacagaga caagccaatg ccacaagtac aggacagatc 2100cgaaaatcat gaccaaaccc ttcaaacaca gtccagggtt ttgactccta tcagcgagga 2160agcagacccc agcgaccaca acgatggtga caatgaaagc attcctcccc tggaatcaga 2220cgacgagggt agcactgata ctactgcagc agaaacaaag cctgccactg cacctcccgc 2280tcccgtctac cgaagtatct ccgtagatga ttctgtcccc tcagagaaca ttcccgcaca 2340gtccaatcaa acgaacaatg aggacaatgt caggaacaat gctcagtcgg agcaatccat 2400tgcagaaatg tatcaacata tcttgaaaac acaaggacct tttgatgcca tcctttacta 2460ccatatgatg aaagaagagc ccatcatttt cagcactagt gatgggaagg agtatacata 2520tccagactct cttgaagatg agtatccacc ctggctcagc gagaaggaag ccatgaacga 2580agacaataga ttcataacca tggatggtca gcagttttac tggcctgtga tgaatcatag 2640aaataaattc atggcaatcc tccagcatca caggtgatcc gacctctaaa actgagctcc 2700taactacaag ctaccccatc actctgccgg aatgccagaa cctccctcca aaacagctcc 2760acatcgagaa cctccgacgc ggtacacagg caagacaggc aacctaatga tgttcctgtt 2820cacccacaac cgcaaccaac acttgatcga cttccaagac aactacaacc cccttagcca 2880actccaccac agaagcacca ccccataaca acaaccccaa accaacaaca ctgcatgtaa 2940gtattgtctc accccaagat gatccctgga caccaacaac cccctaacct ccccaagttg 3000tcattaagaa aaaatatatg atgaagatta aaaccttcat cagagctatt tcttctacgc 3060ttggttagga ccagtattca caaactattt tacaatccct acccaatatg acctctaaca 3120gagcaagggt gacttacaac ccaccaccaa caaccacagg cacacgatcg tgtgggccgg 3180aactttccgg gtggatctct gagcaattga tgacaggcaa gattccgatt accgatatct 3240tcaatgaaat tgaaacctta cctagtataa gtccctcgat ccactccaaa atcaaaaccc 3300caagtgttca aacacgcagt gtccagaccc aaactgaccc aaattgtaat catgattttg 3360cagaggttgt gaaaatgcta acatctctaa cccttgtcgt acaaaaacaa acccttgcaa 3420ctgaatcact tgagcaacgc attactgacc tggaaggtag cctgaaacca gtgtctgaga 3480tcaccaagat tgtttctgca ctaaatagat cctgtgcaga gatggtggcc aaatatgatc 3540ttctagtaat gacgactggt cgtgcaactg ccactgctgc agctactgaa gcatactggg 3600cagaacatgg acgtcctcca ccggggccct cattgtacga ggaggatgca atcaggacta 3660aaattggaaa acaaggggat atggtaccca aggaagtgca agaggccttc cgtaatctgg 3720atagtactgc ccttctaacg gaagagaatt ttgggaaacc agacatatcc gcaaaagact 3780tgcgcaatat catgtatgat cacctcccag gttttggcac agcatttcat caactagtgc 3840aagttatctg caagttaggg aaggacaatt cctcacttga tgtaattcat gcagaatttc 3900aggccagcct tgctgaagga gactctcctc agtgtgccct gattcagata accaaacgga 3960ttcctatttt ccaagatgca gcaccacccg taatccatat tcggtcacgc ggtgatatac 4020caaaggcgtg tcaaaagagc ctccgccctg ttccaccatc accaaagatt gataggggtt 4080gggtatgcat attccagcta caagacggaa aaacactcgg actcaaaatc taaggtgaac 4140aattgcgcaa cctccacagt cgcctatatt gcttccttcc ggaatcaggg tatgatcgcg 4200taaaaaataa gcttccaaca tattgataca cgatccatat ccataatgcc atctccagga 4260atatgagaac gcaaggccat atcaggaccc gatctcaatt ccaatgcaac ctactgttaa 4320gaataaaata accaatgtcc tctagcctta tatgttctca aaaatacaag tgatgaagat 4380taagaaaaag catcctttac ttgagaggag ctaattcttt atacttcatc taatctttaa 4440gtaagttgat cactaccacc atgaggaggg caattctacc tactgcaccg ccagaataca 4500tagaggctgt ctacccaatg agaacggtta gtactagtat caacagtact gccagtggtc 4560cgaactttcc agcaccggat gtaatgatga gtgatacacc ctccaactca ctccgaccaa 4620ttgctgatga taacatcgat catccaagtc atacaccaac cagtgtttca tcagccttta 4680tactcgaggc aatggtgaat gtgatatcgg ggccgaaggt actaatgaag caaattccta 4740tatggctccc cttgggtgtt gctgatcaaa aaacatatag ttttgactca actacagctg 4800caattatgct cgcatcgtac accatcactc actttggcaa aacctccaat ccgcttgtga 4860gaatcaatcg acttggtcct gggatccccg atcacccgtt gcggcttcta agaataggaa 4920atcaagcctt cttgcaagag tttgtgctgc ctccagttca attgccgcag tatttcactt 4980ttgacctgac ggctctaaag ctgatcactc aacctctccc ggcagcaacc tggacggatg 5040atactccgac cggtcctaca ggaatacttc gtcctggaat ttcctttcat cccaaactga 5100gacctatcct attgccaggg aagaccggga aaagaggatc cagctccgat cttacttctc 5160ctgataaaat acaagcaata atgaactttc tccaagacct caaactcgtg ccgattgatc 5220cagccaagaa cattatgggt attgaagtgc cggaactctt ggtccacaga ctaactggaa 5280agaaaatcac aacaaaaaat ggtcaaccaa taattcctat tcttctacca aagtatattg 5340gcatggatcc catttctcag ggagacctca caatggtcat cactcaagac tgtgacactt 5400gccattctcc tgctagtctt cctccagtca gcgagaaatg agcatgaagt ccgaggctgc 5460ccggcccaca cgacccccag ggccttcgtc cggctaccga accaaccatc cgaccttcat 5520caaaaccaaa aaataccgcc acgcgaaagc taaaatgcag gaccacaatc caaccagcaa 5580caccatccat acacaggtat caattgggct gccgcagcat atagacccaa tagcaagctg 5640ctgtccagaa aatagttccg gaaagtaact caaccatcgc aagcccaatg cagctttcag 5700aaatccgcca gcaacccaac tccactgtac ccccaatatt aacctgaatc gactaaccgc 5760actttaattt gaagtacatt tgttcaatgg gttcattatt aacagtgttg cttttagatt 5820gtacctttgc tcacagatag taaattgtta tggtatcaaa tcttattaag aaaaagaaca 5880cgatgaagat taacgcgacc tagagcgctg ccttcatctc atcaatttaa cttgtcaata 5940gagcaaccta gtttgtgatt actcatcttc cgtagttgac aaacactttg ctggttaatt 6000gtaaatatac cacagtcatc atggttacat caggaattct acaattgccc cgtgaacgct 6060tcagaaaaac atcatttttt gtttgggtaa taatcctatt tcacaaagtt ttccctatcc 6120cattgggcgt agttcacaac aacactctcc aggtaagtga tatagataaa ttggtgtgcc 6180gggataaact ttcctccaca agtcagctga aatcggtcgg gcttaatcta gaaggtaatg 6240gagttgccac agatgtacca acagcaacga agagatgggg attccgagct ggtgttccac 6300ccaaagtggt gaactacgaa gctggggagt gggctgaaaa ctgctacaac ctggacatca 6360agaaagcaga tggtagcgaa tgcctacctg aagcccctga gggtgtaaga ggcttccctc 6420gctgccgtta tgtgcacaag gtttctggaa cagggccgtg ccctgaaggt tacgctttcc 6480acaaagaagg cgctttcttc ctgtatgatc gactggcatc aacaatcatc tatcgaagca 6540ccacgttttc agaaggtgtt gtggctttct tgatcctccc cgaaactaaa aaggactttt 6600tccaatcgcc accactacat gaaccggcca atatgacaac agacccatcc agctactacc 6660acacagtcac acttaattat gtggctgaca attttgggac caatatgact aactttctgt 6720ttcaagtgga tcatctaact tatgtgcaac ttgaaccaag attcacacca caatttcttg 6780tccaactcaa tgagaccatt tatactaatg ggcgtcgcag caacaccaca ggaacactaa 6840tttggaaagt aaatcctact gttgacaccg gcgtaggtga atgggccttc tgggaaaata 6900aaaaaacttc acaaaaaccc tttcaagtga agagctgtct gtcatatttg taccaagagc 6960ccaggatcca ggcagcaacc agaagacgaa ggtcactccc accagcttcg ccaacaacca 7020aacctccaag aaccacgaag acttggttcc agaggatccc gcttcagtgg ttcaagtgcg 7080agacctccag agggaaaaca cagtgccgac cccaccccca gacacagtcc ccacaactct 7140gatccccgac acaatggagg aacaaaccac cagccactac gaaccaccaa acatttccag 7200aaaccatcaa gagaggaaca acaccgcaca ccccgaaact ctcgccaaca atcccccaga 7260caacacaacc ccgtcgacac cacctcaaga cggtgagcgg acaagttccc acacaacacc 7320ctccccccgc ccagtcccaa ccagcacaat ccatcccacc acacgagaga ctcacattcc 7380caccacaatg acaacaagcc atgacaccga cagcaatcga cccaacccaa ttgacatcag 7440cgagtctaca gagccaggac cactcaccaa caccacaaga ggggctgcaa atctgctgac 7500aggctcaaga agaacccgaa gggaaatcac cctgagaaca caagccaaat gcaacccaaa 7560cctacactat tggacaaccc aagatgaagg ggctgccatt ggtttagcct ggatacctta 7620cttcgggccc gcagcagagg gaatttatac ggaagggata atgcacaatc aaaatgggct 7680aatttgcggg ttgaggcagc tagcaaatga gacgactcaa gccctacagt tattcttgcg 7740tgctaccacg gaattgcgca ctttctctat attgaatcga aaagccatcg actttttact 7800ccaaagatgg ggaggaacgt gccacatctt aggcccagat tgctgtattg agccccatga 7860ttggactaag aacattactg acaaaataga tcaaatcatt catgatttca ttgataaacc 7920tctaccagat caaacagata atgacaattg gtggacaggg tggaggcaat gggttcctgc 7980cgggatcggg atcacggggg taataatcgc agttatagca ctgctgtgta tttgcaaatt 8040tctactctaa tctagtccga ctctgtacca gcataatggc ctctaaaata agcttttgct 8100tctgcttcct atagttaata catttcagca aaaatcaact attaagtcaa aagaagatcc 8160ctctaataat cctaattacc ttcaaaaatc tagaacttta ttaattctca gggtatttag 8220aacagccaga tgacttgact aagtttgtac tgtaataaaa agatacttga tgaagattaa 8280gaaaaagaca gtcttgtgat tgtcactaat cttcatctca aaacatatta ttttaccaga 8340agctactata gcctacctcc ttgacacata gcaaacctta ctcatgttga taattgtttg 8400cctgctattt acatatttac taacttacaa aattatcttg gggatttctc tgaacatata 8460atcagaattg gcatttaaaa cacaagttag tcctaatgga ctcatttcat gagagagggc 8520gtagcagaac tattcgacag agtgcaagag atgggccgag tcatcaagta agaacaagat 8580catcctccag agacagccac cgcagcgaat atcatacacc taggagctct tcccaagttc 8640gagtcccgac tgtgtttcat cggaagcgta ctgattcttt gacagttcca ccagcaccaa 8700aggacatatg tcctacctta aggaaaggat ttttgtgtga cagcaatttt tgtaaaaagg 8760accatcaact agaaagttta acagataggg agctgctttt gctgattgca cggaaaacct 8820gcggctccct tgaacaacaa ttgaacatca ctgctcctaa agatacacga ttagcaaatc 8880caattgcaga tgatttccaa caaaaagacg gcccaaaaat tacactattg acacttttgg 8940agactgcgga gtattggtca aaacaagata tcaagggcat tgatgactca agactaagag 9000cattactaac cctttgtgcc gtcatgacga ggaaattctc aaaatcccag cttagtctat 9060tgtgtgagag tcatctacga cgagaagggc taggacagga tcaatcagaa tctgttcttg 9120aagtgtatca gcgcttacat agcgacaaag gcggaaattt tgaggcagcc ctatggcaac 9180aatgggaccg acagtccttg atcatgttta taacagcatt tcttaatatt gctttacaat 9240taccctgtga aagttcatct gttgttattt caggattaag gctgctagtg cctcaatcag 9300aagataccga gacctcaacc tacaccgaga cacgtgcatg gtcagaggaa ggtggccccc 9360attaacatct tccacagtcg aatctaccat aatttcccta ttcaacgcag ataagaatca 9420gtactaaacc acaagtgcaa aaattaacaa aacaccagca taagtgaaat cctgtctgtg 9480attagcaaca cgaatgatct tcaatcctgt tgcaattcgc cagtgataat tgtattcaca 9540ttgtggccac aatatactgt cttttcccat tgaaaaataa ggctgaatct attacgctac 9600acaaacttac aggattagca ccacgacggc tcaatactat acctattggt cacggctcga 9660tgtgttaatc acttatattg tattcatttg aaattactca ttaggcaaat actttgatta 9720agaaaaaata attggaaaac cagaaaatcc ctaggtattt aaattcctat ctccggagat 9780ccgagataat taatcaagca atgagggaac aatggtgaac aacaacatat tgttgccccc 9840tttagattgg tcagttccaa aaacaagtga tgaagattaa tgcagatgtc caaggaacac 9900atatttgtga tttaaacgtt ccagttagac tctgttcaag gatcttcatc ttttgtagct 9960ccactctgag tcacaacata attgagtttt tgctcagaac agttatcagg attaaattct 10020ctcaaataac tgaaactact agcatcactc tcaatttcat tacttacgac aatcattatc 10080ttaataatat ttctctaaat tactgactta attagcttgt aatcagataa tatcgaaacc 10140aatttatcat aaggcataat ttgtataagt gatttaggat ttaccccaga agtgaaataa 10200ttcttagaat aaaagaccga ctagaatatc cttaaggctg tctaacgtgc cacacagcta 10260gggttagcct gacatctgga acaagatcga tactaatata gggatttgtt tcatactagc 10320tctctgcaaa cacaatggct aaggcaacag gtaggtacaa cttggtttca cctaaaaagg 10380acctcgagag ggggcttgtt ttgagtgatt tgtgcacgtt tttagttgat cagactatcc 10440aggggtggcg ggtgacttgg gttgggattg aatttgacat cgcccagaaa gggatggctc 10500tactgcatcg gttaaaaact gctgacttcg ctcctgcatg gtcgatgaca aggaatttat 10560ttcctcattt atttcaaaat tcaaattcta ctattgagtc tcccctctgg gcattacgag 10620tgattctggc agctggtatt caagaccagt taattgacca atccttggta gaaccgttgg 10680ccggagccct gagcttagtc tccgattggc ttcttacaac aaacacaaac cattttcaaa 10740tgcgcacgca gcacgctaaa gagcaactga gcttgaagat gctatcatta gtgcgctcta 10800atatcttgaa attcatcagt caattggacg cactacatgt cgtgaactac aatggactct 10860tgagcagtat cgaaattggc actagaaatc ataccattat catcacaaga accaacatgg 10920gtttcctggt agaattacag gagcctgata aatctgccat gaatcaaaag aaaccaggac 10980cagtcaagtt ctccctcctg catgaatcaa ccttcaaggc tctaatcaaa aaacccgcaa 11040ctaagatgca ggccttgatt ctggaattta acagctccct ggcaatatag tccaacgcta 11100ccaaccatca ttttttgtaa ctgcatctct tttatttcct ttctaacttg atacaattat 11160aatcaagatc cctaatccct tttgacgaag tgggctaatt tttgctcatt ctaataataa 11220atcataacct gaataaaaga caccacaata ttataaccca ataacaccta gagaatttct 11280gaattgctaa agattatata ctcgcactaa gagacaagtt aatcaatctt tacttaataa 11340tatactaaat gctagatagc tctggctaac taacctgagt tgtggattac tccttttaaa 11400agtctatcaa tttaagctta tcactaatat taaggaggac tttttaaata agagcaagtg 11460ttatgtagtc ttactaagaa tgatttgagg aagattaaga aaaagtgctt gtggggtctt 11520tccgttgtag aggacacacg agcaaacttc ttcctctaat tttaatatgg caactcaaca 11580tacacaatat ccagatgcaa gattatcttc acccattgtc ttagatcaat gtgatcttgt 11640cacccgtgct tgcggtctgt attcttcata ctcattaaat cctcagttga aaaattgtag 11700actaccaaaa catatttacc gcctcaaatt tgatgctacg gttacaaaat ttttaagcga 11760tgttccaata gttacattgc cgatagatta cttgacccct ttacttttac gaactttatc 11820cggggagggc ttatgccctg tcgaaccaaa gtgcagccaa ttcttagatg aaatagtaag 11880ttatgttttg caggatgcac gttttttaag atactatttt aggcatgttg gagtacacga 11940tgacaatgtt ggaaaaaatt ttgagccaaa gattaaggct ttgatttatg ataatgaatt 12000tctgcaacaa ttgttttatt ggtacgattt agcaatccta acgcgtagag ggcgcctgaa 12060tcgagggaat aaccgttcaa catggtttgc aaatgacgat ttaatagaca ttctcgggta 12120cggtgattat attttctgga aaataccgtt gtcattgttg tcactcaaca cagaggggat 12180tcctcatgca gctaaggact ggtatcacgc atcaatcttc aaagaagcgg ttcaaggtca 12240cacacatatc gtgtcagttt ccactgcaga tgttttaatt atgtgtaagg acatcataac 12300ctgtcgtttc aataccacac tcattgcagc attggcaaat ttagaagatt ctatctgttc 12360tgactatcca caacctgaaa caatctctaa tctgtataag gcaggggatt acttaatctc 12420gatactgggt tcagaaggtt ataaggtcat aaagttttta gaaccactat gtttagctaa 12480gatccaattg tgctcaaatt acactgagag gaaagggaga ttccttactc aaatgcattt 12540ggccgttaat cacacacttg aagaacttat tgagggccgg ggattgaagt cacaacaaga 12600ctggaagatg agggaatttc accgaatctt agtaaattta aagtcaacac cacaacaact 12660ctgtgaattg ttttcagtgc aaaagcattg ggggcatcct gtgctacata gcgagaaggc 12720tattcagaaa gtaaagaaac atgcaaccgt aataaaagca ttgcgtcccg taatcatctt 12780tgagacatat tgtgtgttca agtacagcat tgccaaacat tattttgata gccaagggtc 12840atggtatagt gtaatctcag ataaacatct aacaccaggt ttacactctt acattaagag 12900gaaccaattt ccgccactgc ctatgattaa agacttattg tgggaattct atcaccttga 12960tcatcctccc ttattttcca ccaagattat tagtgacttg agtattttca ttaaggatcg 13020cgctaccgca gtggaaaaaa catgttggga tgcagttttc gagcctaatg ttcttggata 13080tagtcctcca aacaagttct caactaagag ggttcctgaa cagtttcttg aacaagaaaa 13140tttctcgatt gatagtgttc tcacttatgc ccagcgcctg gattatctac ttccacaata 13200ccggaatttt tctttctcac ttaaggaaaa agaattaaat gtaggacgag cttttggtaa 13260gctaccttat cctacacgta atgttcaaac tttatgtgaa gccttattgg cagatggatt 13320agctaaagcc tttcctagta acatgatggt tgtaacagag cgtgagcaga aggaaagcct 13380cttgcaccag gcgtcgtggc accacacaag tgacgatttc ggtgagaatg ccactgttag 13440aggcagcagt tttgttaccg acctagaaaa atacaacttg gcatttagat atgagtttac 13500agctccattt attgaatact gtaatcgatg ttatggtgta aaaaatttat tcaattggat 13560gcattatacg ataccgcaat gttatataca tgtaagtgat tattataatc cccctcatgg 13620agtttcgcta gaaaatcggg aagatccccc ggaaggccct agctcttacc gtggtcatct 13680tgggggaatt gagggactcc aacagaaact ctggaccagc atttcatgtg cacaaatctc 13740attagttgag atcaagactg gtttcaaatt gagatctgcg gtaatgggtg ataatcaatg 13800catcacagtt ctttccgtat ttcctctaga gacagattcc aatgagcaag agcatagctc 13860cgaggacaat gctgctcgcg tagcagccag tttagccaaa gtcacgagtg cctgtggcat 13920cttcctaaaa ccagatgaga cttttgtgca ttcaggcttt atttatttcg gtaagaagca 13980atatttaaat ggcgttcaat tgccacaatc actcaagact gctaccagga ttgctccctt 14040gtcagatgca atctttgatg accttcaggg aactctggct agtataggaa cggcatttga 14100gagatctata tccgagacta gacatgtata cccttgccgg gtggttgccg cattccatac 14160attcttctcc gttaggatcc tccaatacca ccaccttggt ttcaacaaag gaaccgatct 14220aggtcaacta tcactaagca aaccgttgga tttcggaact atcactcttg ctttagcggt 14280acctcaagtt ctaggaggtt tatcgttttt aaacccagag aaatgttttt atcgcaacct 14340tggagacccc gtgacctccg gcctattcca acttaggact tacctgcaaa tgatcaacat 14400ggacgactta tttctacctt taattgccaa gaaccccggg aactgtagtg caattgactt 14460tgtactcaac ccaagcggat tgaatgtccc tgggtcacaa gacctaacat cttttttacg 14520tcagatagtg cgtagaacaa tcacattgag tgcaaaaaat aagctaataa acacattgtt 14580tcactcctca gccgatttag aagatgagat ggtatgtaaa tggctacttt cttcaacacc 14640tgtaatgagt cggtttgctg ctgatatatt ctctcgtact ccgagtggga agcgcttgca 14700gatcctaggt tatttagaag ggactagaac cttgctagcc tccaaagtca tcaataacaa 14760tgcagagact cctattttag ataggttgag gaaaatcaca ctgcagagat ggagtttgtg 14820gtttagctac ctagaccact gtgatcaggt tctagcagat gctttaataa aagtttcttg 14880tacagttgat ttggcgcaaa ttttacgtga atatacctgg gcacacatac tagagggaag 14940acagctcatt ggtgcaacac ttccttgcat gttagaacaa tttaatgtgt tttggctcaa

15000atcgtacgaa caatgcccta aatgtgcaaa atctagaaat ccaaaaggag agccatttgt 15060gtcaattgca attaagaaac aagttgtgag tgcatggccg aatcagtcac ggttaaattg 15120gaccattggg gacggtgtac cttacatcgg gtctcgaaca gaggacaaga ttgggcagcc 15180agcaatcaag cctaagtgtc cctctgctgc cttacgtgaa gcaatagagt tgacatctag 15240actaacatgg gttacccaag gtggtgccaa tagtgatttg ctagttaaac cttttgtaga 15300ggcacgagta aacctgagtg tgcaggagat ccttcaaatg acgccttctc attattcagg 15360gaacatcgta catcggtata atgaccaata cagccctcat tctttcatgg caaatagaat 15420gagtaattcc gcgacgagat tggtggtgtc gacaaatact ctcggggagt tctcaggtgg 15480ggggcaatca gcaagggaca gcaatatcat ctttcaaaat gtaatcaatt tttcggttgc 15540cctatttgat ttacgatttc ggaacaccga aacatcctcc attcagcata atcgtgccca 15600tctccatctt tcacagtgtt gcacacggga agtcccagct caatacctaa cctacacgtc 15660tacgctttcc ttggatctca caaggtaccg agagaatgag ttaatttatg ataacaatcc 15720gttaaaaggt ggacttaatt gcaacctatc ctttgataat ccacttttca agggccaaag 15780gctcaatatc atagaggagg atttgattag atttcctcat ctatctgggt gggaacttgc 15840gaaaaccatc attcagtcca ttatctcaga cagcaataac tcatccacag accccattag 15900cagtggagaa acacgatcat tcacaactca ctttctcaca tatcctaagg ttgggctcct 15960ctatagtttc ggcgccatcg tgagttatta cttagggaat accattatta ggaccaaaaa 16020gctagacctc agtcatttta tgtattactt aacaactcaa atccataatt tgccacatcg 16080ctcgttgagg atacttaagc ccacctttaa acatgttagt gtgatatcaa gactaatgag 16140tattgatcct catttttcaa tctacatcgg gggtacggca ggtgatcgag ggctttcgga 16200tgctaccaga ctattccttc gagtggccat ttcttccttc cttcaattta tcaaaaaatg 16260gatcgtggaa tacaagacag ctattcctct gtgggttata taccctttgg agggacaaaa 16320tccagatcca attaatagct ttctacatct gattatagcc ttactgcaaa atgaatcccc 16380tcaaaacaac atccaattcc aagaagacag aaataatcaa cagttgtccg ataatctagt 16440ttacatgtgc aagagcactg ccagtaattt cttccatgca tcacttgcct attggaggag 16500ccggcacaaa ggacggccca aaaatcgatc gaccgaagaa cagacagtta aacccatacc 16560atatgataat tttcattctg ttaaatgtgc ctcaaaccca ccaagcatcc ccaaatctaa 16620gtcaggaact caaggttcaa gcgcattttt tgagaaactt gaatatgata aagaaagaga 16680attgccaaca gcttccacac cagccgaaca atccaagacc tatatcaagg ccctatccag 16740ccgaatttat catggtaaaa caccatccaa tgccgcaaaa gatgattcaa caacctccaa 16800gggctgcgat tccaaagaag aaaatgccgt tcaagcttca caccgaattg tcctaccatt 16860ttttacattg tcacagaacg actacagaac tccctcagct aaaaagtcag agtatataac 16920tgaaatcacc aaactaattc gacaattaaa ggcaattcca gataccactg tatactgtcg 16980ctttacaggg gttgtatctt caatgcatta taagcttgat gaggttctct gggaattcga 17040tagtttcaaa actgctgtga ctctagctga aggagaaggg tcaggtgcct tattactact 17100acaaaaatat aaggtcagaa caatcttttt taacacttta gctacagagc atagcatcga 17160ggcagaaata gtttctggga caaccacacc tcgaatgctc cttcctgtaa tggccaaact 17220tcatgatgat caaataaatg taatattaaa caattctgct agccaggtta ctgatatcac 17280taaccctgca tggttcactg accagaaatc tagaatcccc acacaagttg agattatgac 17340tatggatgct gaaacgacag aaaatattaa tcggtcaaaa ttatatgagg ctattcagca 17400attaattgtt tcacacattg atacaagggt gctaaagatt gttattataa aggttttttt 17460aagtgatatt gaaggtctcc tgtggcttaa tgaccatctt gcccctttat tcggatccgg 17520ctatttaatt aaacctatta cttcgagtcc aaagtcaagc gaatggtact tatgtctttc 17580aaatttcctt tcagcctctc gacgacggcc tcatcagggt catgctacct gtatgcaagt 17640catccaaaca gcgctacgac tccaagttca aaggagttca tactggctta gccatttagt 17700gcaatatgct gatattaatt tgcacttgag ttatgttaat ttgggtttcc cttcattgga 17760aaaggttctt taccatcgat ataacctagt tgattcacgg aagggtccac tggtctcgat 17820cctttaccat ttaacacact tgcaagcaga gattagagaa ttagtgtgtg actataatca 17880gcaacgacaa agtcgaaccc aaacatacca cttcatcaaa acgacaaagg gccggattac 17940aaaattagtc aatgactacc ttaaatttta tctcgtagtg caagcactga agcataattg 18000tctttggcag gaagaactca gaacacttcc tgacttaatc aatgtttgca atcgatttta 18060ccatataagg gactgctcat gtgaagatcg atttttaatt caaactcttt acttaacccg 18120tatgcaagac tcagaagcaa aattaatgga gagattaacc gggtttctag gattgtatcc 18180taatggtatt aacgcttaag atccccttag aggcatcgca atatgactcc aaacattaaa 18240tgatattgct gtcaatacat ctacctgacc gagagcaagg tttattataa aaaacctata 18300cacatgactg caatgcgtaa tttataccga aacacagtga gggctgcaca tgcaggttcc 18360tgttgagctt taaaagatca tgcaatataa aatgatattt gtatactaat catgttagta 18420ctaactaaca gtactcactg catatactct atcaattaag aaaaattact gtggtttatg 18480catttaaatg acatcacaga tggatataat atagttaatt cttacctaaa tgttgagtta 18540tagtaatttg aagttataat tatgattagt gcttatacta taaataatag ctataccaag 18600tatacacaag aagttatgat tttgtattca aattatattc acaggaactt gtgattaata 18660ataaaagtct cagttgttgg ttgttgagtt gtaaaactcc cgttaaaaat ttattttcca 18720cttataacta ataataatca tagatcagta tgagttgagg ctattcaaac cttagaaaaa 18780ttgtgcgatg ttttttacca tgtcaatctt gatttcaatg atattggagg gcttgtcgat 18840aaattcagta attaacatta agtcagtgtg gaacctcatt ggatatttga tcgtacacaa 18900aatatcttta caaaattgtt ttctcttttt tgtgtgtcca 1894022210PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus L viral protein 2Met Ala Thr Gln His Thr Gln Tyr Pro Asp Ala Arg Leu Ser Ser Pro1 5 10 15Ile Val Leu Asp Gln Cys Asp Leu Val Thr Arg Ala Cys Gly Leu Tyr 20 25 30Ser Ser Tyr Ser Leu Asn Pro Gln Leu Lys Asn Cys Arg Leu Pro Lys 35 40 45His Ile Tyr Arg Leu Lys Phe Asp Ala Thr Val Thr Lys Phe Leu Ser 50 55 60Asp Val Pro Ile Val Thr Leu Pro Ile Asp Tyr Leu Thr Pro Leu Leu65 70 75 80Leu Arg Thr Leu Ser Gly Glu Gly Leu Cys Pro Val Glu Pro Lys Cys 85 90 95Ser Gln Phe Leu Asp Glu Ile Val Ser Tyr Val Leu Gln Asp Ala Arg 100 105 110Phe Leu Arg Tyr Tyr Phe Arg His Val Gly Val His Asp Asp Asn Val 115 120 125Gly Lys Asn Phe Glu Pro Lys Ile Lys Ala Leu Ile Tyr Asp Asn Glu 130 135 140Phe Leu Gln Gln Leu Phe Tyr Trp Tyr Asp Leu Ala Ile Leu Thr Arg145 150 155 160Arg Gly Arg Leu Asn Arg Gly Asn Asn Arg Ser Thr Trp Phe Ala Asn 165 170 175Asp Asp Leu Ile Asp Ile Leu Gly Tyr Gly Asp Tyr Ile Phe Trp Lys 180 185 190Ile Pro Leu Ser Leu Leu Ser Leu Asn Thr Glu Gly Ile Pro His Ala 195 200 205Ala Lys Asp Trp Tyr His Ala Ser Ile Phe Lys Glu Ala Val Gln Gly 210 215 220His Thr His Ile Val Ser Val Ser Thr Ala Asp Val Leu Ile Met Cys225 230 235 240Lys Asp Ile Ile Thr Cys Arg Phe Asn Thr Thr Leu Ile Ala Ala Leu 245 250 255Ala Asn Leu Glu Asp Ser Ile Cys Ser Asp Tyr Pro Gln Pro Glu Thr 260 265 270Ile Ser Asn Leu Tyr Lys Ala Gly Asp Tyr Leu Ile Ser Ile Leu Gly 275 280 285Ser Glu Gly Tyr Lys Val Ile Lys Phe Leu Glu Pro Leu Cys Leu Ala 290 295 300Lys Ile Gln Leu Cys Ser Asn Tyr Thr Glu Arg Lys Gly Arg Phe Leu305 310 315 320Thr Gln Met His Leu Ala Val Asn His Thr Leu Glu Glu Leu Ile Glu 325 330 335Gly Arg Gly Leu Lys Ser Gln Gln Asp Trp Lys Met Arg Glu Phe His 340 345 350Arg Ile Leu Val Asn Leu Lys Ser Thr Pro Gln Gln Leu Cys Glu Leu 355 360 365Phe Ser Val Gln Lys His Trp Gly His Pro Val Leu His Ser Glu Lys 370 375 380Ala Ile Gln Lys Val Lys Lys His Ala Thr Val Ile Lys Ala Leu Arg385 390 395 400Pro Val Ile Ile Phe Glu Thr Tyr Cys Val Phe Lys Tyr Ser Ile Ala 405 410 415Lys His Tyr Phe Asp Ser Gln Gly Ser Trp Tyr Ser Val Ile Ser Asp 420 425 430Lys His Leu Thr Pro Gly Leu His Ser Tyr Ile Lys Arg Asn Gln Phe 435 440 445Pro Pro Leu Pro Met Ile Lys Asp Leu Leu Trp Glu Phe Tyr His Leu 450 455 460Asp His Pro Pro Leu Phe Ser Thr Lys Ile Ile Ser Asp Leu Ser Ile465 470 475 480Phe Ile Lys Asp Arg Ala Thr Ala Val Glu Lys Thr Cys Trp Asp Ala 485 490 495Val Phe Glu Pro Asn Val Leu Gly Tyr Ser Pro Pro Asn Lys Phe Ser 500 505 510Thr Lys Arg Val Pro Glu Gln Phe Leu Glu Gln Glu Asn Phe Ser Ile 515 520 525Asp Ser Val Leu Thr Tyr Ala Gln Arg Leu Asp Tyr Leu Leu Pro Gln 530 535 540Tyr Arg Asn Phe Ser Phe Ser Leu Lys Glu Lys Glu Leu Asn Val Gly545 550 555 560Arg Ala Phe Gly Lys Leu Pro Tyr Pro Thr Arg Asn Val Gln Thr Leu 565 570 575Cys Glu Ala Leu Leu Ala Asp Gly Leu Ala Lys Ala Phe Pro Ser Asn 580 585 590Met Met Val Val Thr Glu Arg Glu Gln Lys Glu Ser Leu Leu His Gln 595 600 605Ala Ser Trp His His Thr Ser Asp Asp Phe Gly Glu Asn Ala Thr Val 610 615 620Arg Gly Ser Ser Phe Val Thr Asp Leu Glu Lys Tyr Asn Leu Ala Phe625 630 635 640Arg Tyr Glu Phe Thr Ala Pro Phe Ile Glu Tyr Cys Asn Arg Cys Tyr 645 650 655Gly Val Lys Asn Leu Phe Asn Trp Met His Tyr Thr Ile Pro Gln Cys 660 665 670Tyr Ile His Val Ser Asp Tyr Tyr Asn Pro Pro His Gly Val Ser Leu 675 680 685Glu Asn Arg Glu Asp Pro Pro Glu Gly Pro Ser Ser Tyr Arg Gly His 690 695 700Leu Gly Gly Ile Glu Gly Leu Gln Gln Lys Leu Trp Thr Ser Ile Ser705 710 715 720Cys Ala Gln Ile Ser Leu Val Glu Ile Lys Thr Gly Phe Lys Leu Arg 725 730 735Ser Ala Val Met Gly Asp Asn Gln Cys Ile Thr Val Leu Ser Val Phe 740 745 750Pro Leu Glu Thr Asp Ser Asn Glu Gln Glu His Ser Ser Glu Asp Asn 755 760 765Ala Ala Arg Val Ala Ala Ser Leu Ala Lys Val Thr Ser Ala Cys Gly 770 775 780Ile Phe Leu Lys Pro Asp Glu Thr Phe Val His Ser Gly Phe Ile Tyr785 790 795 800Phe Gly Lys Lys Gln Tyr Leu Asn Gly Val Gln Leu Pro Gln Ser Leu 805 810 815Lys Thr Ala Thr Arg Ile Ala Pro Leu Ser Asp Ala Ile Phe Asp Asp 820 825 830Leu Gln Gly Thr Leu Ala Ser Ile Gly Thr Ala Phe Glu Arg Ser Ile 835 840 845Ser Glu Thr Arg His Val Tyr Pro Cys Arg Val Val Ala Ala Phe His 850 855 860Thr Phe Phe Ser Val Arg Ile Leu Gln Tyr His His Leu Gly Phe Asn865 870 875 880Lys Gly Thr Asp Leu Gly Gln Leu Ser Leu Ser Lys Pro Leu Asp Phe 885 890 895Gly Thr Ile Thr Leu Ala Leu Ala Val Pro Gln Val Leu Gly Gly Leu 900 905 910Ser Phe Leu Asn Pro Glu Lys Cys Phe Tyr Arg Asn Leu Gly Asp Pro 915 920 925Val Thr Ser Gly Leu Phe Gln Leu Arg Thr Tyr Leu Gln Met Ile Asn 930 935 940Met Asp Asp Leu Phe Leu Pro Leu Ile Ala Lys Asn Pro Gly Asn Cys945 950 955 960Ser Ala Ile Asp Phe Val Leu Asn Pro Ser Gly Leu Asn Val Pro Gly 965 970 975Ser Gln Asp Leu Thr Ser Phe Leu Arg Gln Ile Val Arg Arg Thr Ile 980 985 990Thr Leu Ser Ala Lys Asn Lys Leu Ile Asn Thr Leu Phe His Ser Ser 995 1000 1005Ala Asp Leu Glu Asp Glu Met Val Cys Lys Trp Leu Leu Ser Ser 1010 1015 1020Thr Pro Val Met Ser Arg Phe Ala Ala Asp Ile Phe Ser Arg Thr 1025 1030 1035Pro Ser Gly Lys Arg Leu Gln Ile Leu Gly Tyr Leu Glu Gly Thr 1040 1045 1050Arg Thr Leu Leu Ala Ser Lys Val Ile Asn Asn Asn Ala Glu Thr 1055 1060 1065Pro Ile Leu Asp Arg Leu Arg Lys Ile Thr Leu Gln Arg Trp Ser 1070 1075 1080Leu Trp Phe Ser Tyr Leu Asp His Cys Asp Gln Val Leu Ala Asp 1085 1090 1095Ala Leu Ile Lys Val Ser Cys Thr Val Asp Leu Ala Gln Ile Leu 1100 1105 1110Arg Glu Tyr Thr Trp Ala His Ile Leu Glu Gly Arg Gln Leu Ile 1115 1120 1125Gly Ala Thr Leu Pro Cys Met Leu Glu Gln Phe Asn Val Phe Trp 1130 1135 1140Leu Lys Ser Tyr Glu Gln Cys Pro Lys Cys Ala Lys Ser Arg Asn 1145 1150 1155Pro Lys Gly Glu Pro Phe Val Ser Ile Ala Ile Lys Lys Gln Val 1160 1165 1170Val Ser Ala Trp Pro Asn Gln Ser Arg Leu Asn Trp Thr Ile Gly 1175 1180 1185Asp Gly Val Pro Tyr Ile Gly Ser Arg Thr Glu Asp Lys Ile Gly 1190 1195 1200Gln Pro Ala Ile Lys Pro Lys Cys Pro Ser Ala Ala Leu Arg Glu 1205 1210 1215Ala Ile Glu Leu Thr Ser Arg Leu Thr Trp Val Thr Gln Gly Gly 1220 1225 1230Ala Asn Ser Asp Leu Leu Val Lys Pro Phe Val Glu Ala Arg Val 1235 1240 1245Asn Leu Ser Val Gln Glu Ile Leu Gln Met Thr Pro Ser His Tyr 1250 1255 1260Ser Gly Asn Ile Val His Arg Tyr Asn Asp Gln Tyr Ser Pro His 1265 1270 1275Ser Phe Met Ala Asn Arg Met Ser Asn Ser Ala Thr Arg Leu Val 1280 1285 1290Val Ser Thr Asn Thr Leu Gly Glu Phe Ser Gly Gly Gly Gln Ser 1295 1300 1305Ala Arg Asp Ser Asn Ile Ile Phe Gln Asn Val Ile Asn Phe Ser 1310 1315 1320Val Ala Leu Phe Asp Leu Arg Phe Arg Asn Thr Glu Thr Ser Ser 1325 1330 1335Ile Gln His Asn Arg Ala His Leu His Leu Ser Gln Cys Cys Thr 1340 1345 1350Arg Glu Val Pro Ala Gln Tyr Leu Thr Tyr Thr Ser Thr Leu Ser 1355 1360 1365Leu Asp Leu Thr Arg Tyr Arg Glu Asn Glu Leu Ile Tyr Asp Asn 1370 1375 1380Asn Pro Leu Lys Gly Gly Leu Asn Cys Asn Leu Ser Phe Asp Asn 1385 1390 1395Pro Leu Phe Lys Gly Gln Arg Leu Asn Ile Ile Glu Glu Asp Leu 1400 1405 1410Ile Arg Phe Pro His Leu Ser Gly Trp Glu Leu Ala Lys Thr Ile 1415 1420 1425Ile Gln Ser Ile Ile Ser Asp Ser Asn Asn Ser Ser Thr Asp Pro 1430 1435 1440Ile Ser Ser Gly Glu Thr Arg Ser Phe Thr Thr His Phe Leu Thr 1445 1450 1455Tyr Pro Lys Val Gly Leu Leu Tyr Ser Phe Gly Ala Ile Val Ser 1460 1465 1470Tyr Tyr Leu Gly Asn Thr Ile Ile Arg Thr Lys Lys Leu Asp Leu 1475 1480 1485Ser His Phe Met Tyr Tyr Leu Thr Thr Gln Ile His Asn Leu Pro 1490 1495 1500His Arg Ser Leu Arg Ile Leu Lys Pro Thr Phe Lys His Val Ser 1505 1510 1515Val Ile Ser Arg Leu Met Ser Ile Asp Pro His Phe Ser Ile Tyr 1520 1525 1530Ile Gly Gly Thr Ala Gly Asp Arg Gly Leu Ser Asp Ala Thr Arg 1535 1540 1545Leu Phe Leu Arg Val Ala Ile Ser Ser Phe Leu Gln Phe Ile Lys 1550 1555 1560Lys Trp Ile Val Glu Tyr Lys Thr Ala Ile Pro Leu Trp Val Ile 1565 1570 1575Tyr Pro Leu Glu Gly Gln Asn Pro Asp Pro Ile Asn Ser Phe Leu 1580 1585 1590His Leu Ile Ile Ala Leu Leu Gln Asn Glu Ser Pro Gln Asn Asn 1595 1600 1605Ile Gln Phe Gln Glu Asp Arg Asn Asn Gln Gln Leu Ser Asp Asn 1610 1615 1620Leu Val Tyr Met Cys Lys Ser Thr Ala Ser Asn Phe Phe His Ala 1625 1630 1635Ser Leu Ala Tyr Trp Arg Ser Arg His Lys Gly Arg Pro Lys Asn 1640 1645 1650Arg Ser Thr Glu Glu Gln Thr Val Lys Pro Ile Pro Tyr Asp Asn 1655 1660 1665Phe His Ser Val Lys Cys Ala Ser Asn Pro Pro Ser Ile Pro Lys 1670 1675 1680Ser Lys Ser Gly Thr Gln Gly Ser Ser Ala Phe Phe Glu Lys Leu 1685 1690 1695Glu Tyr Asp Lys Glu Arg Glu Leu Pro Thr Ala Ser Thr Pro Ala 1700 1705 1710Glu Gln Ser Lys Thr Tyr Ile Lys Ala Leu Ser Ser Arg Ile Tyr 1715 1720 1725His Gly Lys Thr Pro Ser Asn Ala Ala Lys Asp Asp Ser Thr Thr 1730 1735 1740Ser Lys Gly Cys Asp Ser Lys Glu Glu Asn Ala Val Gln Ala Ser 1745 1750 1755His Arg Ile Val Leu Pro Phe Phe Thr Leu Ser Gln Asn Asp Tyr 1760 1765 1770Arg Thr Pro Ser Ala Lys Lys Ser Glu Tyr Ile Thr Glu Ile Thr 1775 1780 1785Lys Leu Ile Arg Gln Leu Lys Ala Ile Pro Asp Thr Thr Val Tyr 1790

1795 1800Cys Arg Phe Thr Gly Val Val Ser Ser Met His Tyr Lys Leu Asp 1805 1810 1815Glu Val Leu Trp Glu Phe Asp Ser Phe Lys Thr Ala Val Thr Leu 1820 1825 1830Ala Glu Gly Glu Gly Ser Gly Ala Leu Leu Leu Leu Gln Lys Tyr 1835 1840 1845Lys Val Arg Thr Ile Phe Phe Asn Thr Leu Ala Thr Glu His Ser 1850 1855 1860Ile Glu Ala Glu Ile Val Ser Gly Thr Thr Thr Pro Arg Met Leu 1865 1870 1875Leu Pro Val Met Ala Lys Leu His Asp Asp Gln Ile Asn Val Ile 1880 1885 1890Leu Asn Asn Ser Ala Ser Gln Val Thr Asp Ile Thr Asn Pro Ala 1895 1900 1905Trp Phe Thr Asp Gln Lys Ser Arg Ile Pro Thr Gln Val Glu Ile 1910 1915 1920Met Thr Met Asp Ala Glu Thr Thr Glu Asn Ile Asn Arg Ser Lys 1925 1930 1935Leu Tyr Glu Ala Ile Gln Gln Leu Ile Val Ser His Ile Asp Thr 1940 1945 1950Arg Val Leu Lys Ile Val Ile Ile Lys Val Phe Leu Ser Asp Ile 1955 1960 1965Glu Gly Leu Leu Trp Leu Asn Asp His Leu Ala Pro Leu Phe Gly 1970 1975 1980Ser Gly Tyr Leu Ile Lys Pro Ile Thr Ser Ser Pro Lys Ser Ser 1985 1990 1995Glu Trp Tyr Leu Cys Leu Ser Asn Phe Leu Ser Ala Ser Arg Arg 2000 2005 2010Arg Pro His Gln Gly His Ala Thr Cys Met Gln Val Ile Gln Thr 2015 2020 2025Ala Leu Arg Leu Gln Val Gln Arg Ser Ser Tyr Trp Leu Ser His 2030 2035 2040Leu Val Gln Tyr Ala Asp Ile Asn Leu His Leu Ser Tyr Val Asn 2045 2050 2055Leu Gly Phe Pro Ser Leu Glu Lys Val Leu Tyr His Arg Tyr Asn 2060 2065 2070Leu Val Asp Ser Arg Lys Gly Pro Leu Val Ser Ile Leu Tyr His 2075 2080 2085Leu Thr His Leu Gln Ala Glu Ile Arg Glu Leu Val Cys Asp Tyr 2090 2095 2100Asn Gln Gln Arg Gln Ser Arg Thr Gln Thr Tyr His Phe Ile Lys 2105 2110 2115Thr Thr Lys Gly Arg Ile Thr Lys Leu Val Asn Asp Tyr Leu Lys 2120 2125 2130Phe Tyr Leu Val Val Gln Ala Leu Lys His Asn Cys Leu Trp Gln 2135 2140 2145Glu Glu Leu Arg Thr Leu Pro Asp Leu Ile Asn Val Cys Asn Arg 2150 2155 2160Phe Tyr His Ile Arg Asp Cys Ser Cys Glu Asp Arg Phe Leu Ile 2165 2170 2175Gln Thr Leu Tyr Leu Thr Arg Met Gln Asp Ser Glu Ala Lys Leu 2180 2185 2190Met Glu Arg Leu Thr Gly Phe Leu Gly Leu Tyr Pro Asn Gly Ile 2195 2200 2205Asn Ala 22103739PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus NP viral protein 3Met Asp Pro Arg Pro Ile Arg Thr Trp Met Met His Asn Thr Ser Glu1 5 10 15Val Glu Ala Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln 20 25 30Gln Gly Ile Val Arg Gln Arg Ile Ile Pro Val Tyr Gln Ile Ser Asn 35 40 45Leu Glu Glu Val Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val 50 55 60Asp Phe Gln Asp Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His65 70 75 80His Ala Tyr Gln Gly Asp Tyr Lys Gln Phe Leu Glu Ser Asn Ala Val 85 90 95Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Met Lys Lys Lys Glu 100 105 110Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Ala Ser Ser Gly Lys 115 120 125Asn Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu 130 135 140Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys145 150 155 160Leu Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile 165 170 175Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ser Trp Gln 180 185 190Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe 195 200 205Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly 210 215 220His Asp Ala Asn Asp Ala Val Ile Ala Asn Ser Val Ala Gln Ala Arg225 230 235 240Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln 245 250 255Lys Thr Glu His Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys 260 265 270Val Lys Asn Glu Val Ser Ser Phe Lys Ala Ala Leu Ala Ser Leu Ala 275 280 285Gln His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295 300Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala305 310 315 320Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val 325 330 335Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys 340 345 350Gln Leu Gln Lys Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu 355 360 365Asp Asp Gln Glu Lys Lys Ile Leu Lys Asp Phe His Gln Lys Lys Asn 370 375 380Glu Ile Ser Phe Gln Gln Thr Thr Ala Met Val Thr Leu Arg Lys Glu385 390 395 400Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ser Thr Ser Ile Leu Lys 405 410 415Thr Gly Arg Arg Tyr Asp Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro 420 425 430Ile Asn Asp Asn Glu Asn Ser Gly Gln Asn Asp Asp Asp Pro Thr Asp 435 440 445Ser Gln Asp Thr Thr Ile Pro Asp Val Ile Ile Asp Pro Asn Asp Gly 450 455 460Gly Tyr Asn Asn Tyr Ser Asp Tyr Ala Asn Asp Ala Ala Ser Ala Pro465 470 475 480Asp Asp Leu Val Leu Phe Asp Leu Glu Asp Glu Asp Asp Ala Asp Asn 485 490 495Pro Ala Gln Asn Thr Pro Glu Lys Asn Asp Arg Pro Ala Thr Thr Lys 500 505 510Leu Arg Asn Gly Gln Asp Gln Asp Gly Asn Gln Gly Glu Thr Ala Ser 515 520 525Pro Arg Val Ala Pro Asn Gln Tyr Arg Asp Lys Pro Met Pro Gln Val 530 535 540Gln Asp Arg Ser Glu Asn His Asp Gln Thr Leu Gln Thr Gln Ser Arg545 550 555 560Val Leu Thr Pro Ile Ser Glu Glu Ala Asp Pro Ser Asp His Asn Asp 565 570 575Gly Asp Asn Glu Ser Ile Pro Pro Leu Glu Ser Asp Asp Glu Gly Ser 580 585 590Thr Asp Thr Thr Ala Ala Glu Thr Lys Pro Ala Thr Ala Pro Pro Ala 595 600 605Pro Val Tyr Arg Ser Ile Ser Val Asp Asp Ser Val Pro Ser Glu Asn 610 615 620Ile Pro Ala Gln Ser Asn Gln Thr Asn Asn Glu Asp Asn Val Arg Asn625 630 635 640Asn Ala Gln Ser Glu Gln Ser Ile Ala Glu Met Tyr Gln His Ile Leu 645 650 655Lys Thr Gln Gly Pro Phe Asp Ala Ile Leu Tyr Tyr His Met Met Lys 660 665 670Glu Glu Pro Ile Ile Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr 675 680 685Pro Asp Ser Leu Glu Asp Glu Tyr Pro Pro Trp Leu Ser Glu Lys Glu 690 695 700Ala Met Asn Glu Asp Asn Arg Phe Ile Thr Met Asp Gly Gln Gln Phe705 710 715 720Tyr Trp Pro Val Met Asn His Arg Asn Lys Phe Met Ala Ile Leu Gln 725 730 735His His Arg4373PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus SGP viral protein 4Met Val Thr Ser Gly Ile Leu Gln Leu Pro Arg Glu Arg Phe Arg Lys1 5 10 15Thr Ser Phe Phe Val Trp Val Ile Ile Leu Phe His Lys Val Phe Pro 20 25 30Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 35 40 45Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65 70 75 80Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 85 90 95Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Asp 100 105 110Ile Lys Lys Ala Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 115 120 125Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Pro Glu Gly Tyr Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Ser Thr Thr Phe 165 170 175Ser Glu Gly Val Val Ala Phe Leu Ile Leu Pro Glu Thr Lys Lys Asp 180 185 190Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 195 200 205Pro Ser Ser Tyr Tyr His Thr Val Thr Leu Asn Tyr Val Ala Asp Asn 210 215 220Phe Gly Thr Asn Met Thr Asn Phe Leu Phe Gln Val Asp His Leu Thr225 230 235 240Tyr Val Gln Leu Glu Pro Arg Phe Thr Pro Gln Phe Leu Val Gln Leu 245 250 255Asn Glu Thr Ile Tyr Thr Asn Gly Arg Arg Ser Asn Thr Thr Gly Thr 260 265 270Leu Ile Trp Lys Val Asn Pro Thr Val Asp Thr Gly Val Gly Glu Trp 275 280 285Ala Phe Trp Glu Asn Lys Lys Thr Ser Gln Lys Pro Phe Gln Val Lys 290 295 300Ser Cys Leu Ser Tyr Leu Tyr Gln Glu Pro Arg Ile Gln Ala Ala Thr305 310 315 320Arg Arg Arg Arg Ser Leu Pro Pro Ala Ser Pro Thr Thr Lys Pro Pro 325 330 335Arg Thr Thr Lys Thr Trp Phe Gln Arg Ile Pro Leu Gln Trp Phe Lys 340 345 350Cys Glu Thr Ser Arg Gly Lys Thr Gln Cys Arg Pro His Pro Gln Thr 355 360 365Gln Ser Pro Gln Leu 3705251PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus VP24 viral protein 5Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu Val Ser Pro Lys Lys Asp1 5 10 15Leu Glu Arg Gly Leu Val Leu Ser Asp Leu Cys Thr Phe Leu Val Asp 20 25 30Gln Thr Ile Gln Gly Trp Arg Val Thr Trp Val Gly Ile Glu Phe Asp 35 40 45Ile Ala Gln Lys Gly Met Ala Leu Leu His Arg Leu Lys Thr Ala Asp 50 55 60Phe Ala Pro Ala Trp Ser Met Thr Arg Asn Leu Phe Pro His Leu Phe65 70 75 80Gln Asn Ser Asn Ser Thr Ile Glu Ser Pro Leu Trp Ala Leu Arg Val 85 90 95Ile Leu Ala Ala Gly Ile Gln Asp Gln Leu Ile Asp Gln Ser Leu Val 100 105 110Glu Pro Leu Ala Gly Ala Leu Ser Leu Val Ser Asp Trp Leu Leu Thr 115 120 125Thr Asn Thr Asn His Phe Gln Met Arg Thr Gln His Ala Lys Glu Gln 130 135 140Leu Ser Leu Lys Met Leu Ser Leu Val Arg Ser Asn Ile Leu Lys Phe145 150 155 160Ile Ser Gln Leu Asp Ala Leu His Val Val Asn Tyr Asn Gly Leu Leu 165 170 175Ser Ser Ile Glu Ile Gly Thr Arg Asn His Thr Ile Ile Ile Thr Arg 180 185 190Thr Asn Met Gly Phe Leu Val Glu Leu Gln Glu Pro Asp Lys Ser Ala 195 200 205Met Asn Gln Lys Lys Pro Gly Pro Val Lys Phe Ser Leu Leu His Glu 210 215 220Ser Thr Phe Lys Ala Leu Ile Lys Lys Pro Ala Thr Lys Met Gln Ala225 230 235 240Leu Ile Leu Glu Phe Asn Ser Ser Leu Ala Ile 245 2506289PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus VP30 viral protein 6Met Asp Ser Phe His Glu Arg Gly Arg Ser Arg Thr Ile Arg Gln Ser1 5 10 15Ala Arg Asp Gly Pro Ser His Gln Val Arg Thr Arg Ser Ser Ser Arg 20 25 30Asp Ser His Arg Ser Glu Tyr His Thr Pro Arg Ser Ser Ser Gln Val 35 40 45Arg Val Pro Thr Val Phe His Arg Lys Arg Thr Asp Ser Leu Thr Val 50 55 60Pro Pro Ala Pro Lys Asp Ile Cys Pro Thr Leu Arg Lys Gly Phe Leu65 70 75 80Cys Asp Ser Asn Phe Cys Lys Lys Asp His Gln Leu Glu Ser Leu Thr 85 90 95Asp Arg Glu Leu Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly Ser Leu 100 105 110Glu Gln Gln Leu Asn Ile Thr Ala Pro Lys Asp Thr Arg Leu Ala Asn 115 120 125Pro Ile Ala Asp Asp Phe Gln Gln Lys Asp Gly Pro Lys Ile Thr Leu 130 135 140Leu Thr Leu Leu Glu Thr Ala Glu Tyr Trp Ser Lys Gln Asp Ile Lys145 150 155 160Gly Ile Asp Asp Ser Arg Leu Arg Ala Leu Leu Thr Leu Cys Ala Val 165 170 175Met Thr Arg Lys Phe Ser Lys Ser Gln Leu Ser Leu Leu Cys Glu Ser 180 185 190His Leu Arg Arg Glu Gly Leu Gly Gln Asp Gln Ser Glu Ser Val Leu 195 200 205Glu Val Tyr Gln Arg Leu His Ser Asp Lys Gly Gly Asn Phe Glu Ala 210 215 220Ala Leu Trp Gln Gln Trp Asp Arg Gln Ser Leu Ile Met Phe Ile Thr225 230 235 240Ala Phe Leu Asn Ile Ala Leu Gln Leu Pro Cys Glu Ser Ser Ser Val 245 250 255Val Ile Ser Gly Leu Arg Leu Leu Val Pro Gln Ser Glu Asp Thr Glu 260 265 270Thr Ser Thr Tyr Thr Glu Thr Arg Ala Trp Ser Glu Glu Gly Gly Pro 275 280 285His 7341PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus VP35 viral protein 7Met Thr Ser Asn Arg Ala Arg Val Thr Tyr Asn Pro Pro Pro Thr Thr1 5 10 15Thr Gly Thr Arg Ser Cys Gly Pro Glu Leu Ser Gly Trp Ile Ser Glu 20 25 30Gln Leu Met Thr Gly Lys Ile Pro Ile Thr Asp Ile Phe Asn Glu Ile 35 40 45Glu Thr Leu Pro Ser Ile Ser Pro Ser Ile His Ser Lys Ile Lys Thr 50 55 60Pro Ser Val Gln Thr Arg Ser Val Gln Thr Gln Thr Asp Pro Asn Cys65 70 75 80Asn His Asp Phe Ala Glu Val Val Lys Met Leu Thr Ser Leu Thr Leu 85 90 95Val Val Gln Lys Gln Thr Leu Ala Thr Glu Ser Leu Glu Gln Arg Ile 100 105 110Thr Asp Leu Glu Gly Ser Leu Lys Pro Val Ser Glu Ile Thr Lys Ile 115 120 125Val Ser Ala Leu Asn Arg Ser Cys Ala Glu Met Val Ala Lys Tyr Asp 130 135 140Leu Leu Val Met Thr Thr Gly Arg Ala Thr Ala Thr Ala Ala Ala Thr145 150 155 160Glu Ala Tyr Trp Ala Glu His Gly Arg Pro Pro Pro Gly Pro Ser Leu 165 170 175Tyr Glu Glu Asp Ala Ile Arg Thr Lys Ile Gly Lys Gln Gly Asp Met 180 185 190Val Pro Lys Glu Val Gln Glu Ala Phe Arg Asn Leu Asp Ser Thr Ala 195 200 205Leu Leu Thr Glu Glu Asn Phe Gly Lys Pro Asp Ile Ser Ala Lys Asp 210 215 220Leu Arg Asn Ile Met Tyr Asp His Leu Pro Gly Phe Gly Thr Ala Phe225 230 235 240His Gln Leu Val Gln Val Ile Cys Lys Leu Gly Lys Asp Asn Ser Ser 245 250 255Leu Asp Val Ile His Ala Glu Phe Gln Ala Ser Leu Ala Glu Gly Asp 260 265 270Ser Pro Gln Cys Ala Leu Ile Gln Ile Thr Lys Arg Ile Pro Ile Phe 275 280 285Gln Asp Ala Ala Pro Pro Val Ile His Ile Arg Ser Arg Gly Asp Ile 290 295 300Pro Lys Ala Cys Gln Lys Ser Leu Arg Pro Val Pro Pro Ser Pro Lys305 310 315 320Ile Asp Arg Gly Trp Val Cys Ile Phe Gln Leu Gln Asp Gly Lys Thr 325 330 335Leu Gly Leu Lys Ile 3408326PRTBundibugyo ebolavirusmisc_featureBundibugyo

ebolavirus VP40 viral protein 8Met Arg Arg Ala Ile Leu Pro Thr Ala Pro Pro Glu Tyr Ile Glu Ala1 5 10 15Val Tyr Pro Met Arg Thr Val Ser Thr Ser Ile Asn Ser Thr Ala Ser 20 25 30Gly Pro Asn Phe Pro Ala Pro Asp Val Met Met Ser Asp Thr Pro Ser 35 40 45Asn Ser Leu Arg Pro Ile Ala Asp Asp Asn Ile Asp His Pro Ser His 50 55 60Thr Pro Thr Ser Val Ser Ser Ala Phe Ile Leu Glu Ala Met Val Asn65 70 75 80Val Ile Ser Gly Pro Lys Val Leu Met Lys Gln Ile Pro Ile Trp Leu 85 90 95Pro Leu Gly Val Ala Asp Gln Lys Thr Tyr Ser Phe Asp Ser Thr Thr 100 105 110Ala Ala Ile Met Leu Ala Ser Tyr Thr Ile Thr His Phe Gly Lys Thr 115 120 125Ser Asn Pro Leu Val Arg Ile Asn Arg Leu Gly Pro Gly Ile Pro Asp 130 135 140His Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu Gln Glu145 150 155 160Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe Asp Leu 165 170 175Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr Trp Thr 180 185 190Asp Asp Thr Pro Thr Gly Pro Thr Gly Ile Leu Arg Pro Gly Ile Ser 195 200 205Phe His Pro Lys Leu Arg Pro Ile Leu Leu Pro Gly Lys Thr Gly Lys 210 215 220Arg Gly Ser Ser Ser Asp Leu Thr Ser Pro Asp Lys Ile Gln Ala Ile225 230 235 240Met Asn Phe Leu Gln Asp Leu Lys Leu Val Pro Ile Asp Pro Ala Lys 245 250 255Asn Ile Met Gly Ile Glu Val Pro Glu Leu Leu Val His Arg Leu Thr 260 265 270Gly Lys Lys Ile Thr Thr Lys Asn Gly Gln Pro Ile Ile Pro Ile Leu 275 280 285Leu Pro Lys Tyr Ile Gly Met Asp Pro Ile Ser Gln Gly Asp Leu Thr 290 295 300Met Val Ile Thr Gln Asp Cys Asp Thr Cys His Ser Pro Ala Ser Leu305 310 315 320Pro Pro Val Ser Glu Lys 3259676PRTBundibugyo ebolavirusmisc_featureBundibugyo ebolavirus GP viral protein 9Met Val Thr Ser Gly Ile Leu Gln Leu Pro Arg Glu Arg Phe Arg Lys1 5 10 15Thr Ser Phe Phe Val Trp Val Ile Ile Leu Phe His Lys Val Phe Pro 20 25 30Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 35 40 45Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65 70 75 80Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 85 90 95Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Asp 100 105 110Ile Lys Lys Ala Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 115 120 125Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Pro Glu Gly Tyr Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Ser Thr Thr Phe 165 170 175Ser Glu Gly Val Val Ala Phe Leu Ile Leu Pro Glu Thr Lys Lys Asp 180 185 190Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 195 200 205Pro Ser Ser Tyr Tyr His Thr Val Thr Leu Asn Tyr Val Ala Asp Asn 210 215 220Phe Gly Thr Asn Met Thr Asn Phe Leu Phe Gln Val Asp His Leu Thr225 230 235 240Tyr Val Gln Leu Glu Pro Arg Phe Thr Pro Gln Phe Leu Val Gln Leu 245 250 255Asn Glu Thr Ile Tyr Thr Asn Gly Arg Arg Ser Asn Thr Thr Gly Thr 260 265 270Leu Ile Trp Lys Val Asn Pro Thr Val Asp Thr Gly Val Gly Glu Trp 275 280 285Ala Phe Trp Glu Asn Lys Lys Asn Phe Thr Lys Thr Leu Ser Ser Glu 290 295 300Glu Leu Ser Val Ile Phe Val Pro Arg Ala Gln Asp Pro Gly Ser Asn305 310 315 320Gln Lys Thr Lys Val Thr Pro Thr Ser Phe Ala Asn Asn Gln Thr Ser 325 330 335Lys Asn His Glu Asp Leu Val Pro Glu Asp Pro Ala Ser Val Val Gln 340 345 350Val Arg Asp Leu Gln Arg Glu Asn Thr Val Pro Thr Pro Pro Pro Asp 355 360 365Thr Val Pro Thr Thr Leu Ile Pro Asp Thr Met Glu Glu Gln Thr Thr 370 375 380Ser His Tyr Glu Pro Pro Asn Ile Ser Arg Asn His Gln Glu Arg Asn385 390 395 400Asn Thr Ala His Pro Glu Thr Leu Ala Asn Asn Pro Pro Asp Asn Thr 405 410 415Thr Pro Ser Thr Pro Pro Gln Asp Gly Glu Arg Thr Ser Ser His Thr 420 425 430Thr Pro Ser Pro Arg Pro Val Pro Thr Ser Thr Ile His Pro Thr Thr 435 440 445Arg Glu Thr His Ile Pro Thr Thr Met Thr Thr Ser His Asp Thr Asp 450 455 460Ser Asn Arg Pro Asn Pro Ile Asp Ile Ser Glu Ser Thr Glu Pro Gly465 470 475 480Pro Leu Thr Asn Thr Thr Arg Gly Ala Ala Asn Leu Leu Thr Gly Ser 485 490 495Arg Arg Thr Arg Arg Glu Ile Thr Leu Arg Thr Gln Ala Lys Cys Asn 500 505 510Pro Asn Leu His Tyr Trp Thr Thr Gln Asp Glu Gly Ala Ala Ile Gly 515 520 525Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Thr 530 535 540Glu Gly Ile Met His Asn Gln Asn Gly Leu Ile Cys Gly Leu Arg Gln545 550 555 560Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr 565 570 575Thr Glu Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile Asp Phe 580 585 590Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys 595 600 605Cys Ile Glu Pro His Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp 610 615 620Gln Ile Ile His Asp Phe Ile Asp Lys Pro Leu Pro Asp Gln Thr Asp625 630 635 640Asn Asp Asn Trp Trp Thr Gly Trp Arg Gln Trp Val Pro Ala Gly Ile 645 650 655Gly Ile Thr Gly Val Ile Ile Ala Val Ile Ala Leu Leu Cys Ile Cys 660 665 670Lys Phe Leu Leu 6751018935DNACote dIvoire ebolavirus 10cggacacaca aaaagaaaga aggttttttg atctttattg tgtgcgaata actatgagga 60agattaataa ttttcctctc attgacactt acattaagat taagattctc attgatctgt 120tacttactct gaggataata attggtgttc agaagtaccc cattccccag tgggggcaaa 180gacagtccaa aagactcaac ttgtcctatt caactaatct gttttgtctc agtagttcac 240atattgatca tacccaggag ttggacctaa ttccaaagct tagagtggga cctagtgtat 300cctcggggct gtaatataat cagccattta acacataaca agccctactg ttttcttgtt 360ttgccgtgca tttagaataa gagacaactt aaacctccga ttcggcaaca cagggaataa 420tctcaccaga cccggcagtg tcttcaggct tcatagcccc aagatggaga gtcgggccca 480caaagcatgg atgacgcaca ccgcatcagg tttcgaaaca gattaccata agattttaac 540agcaggattg tcagtccaac aaggcattgt gagacaacgg gtcattcaag tccaccaggt 600tacaaaccta gaagaaatat gccaattgat cattcaagcc tttgaagctg gtgttgattt 660tcaagagagt gcagacagtt tcttgctgat gctatgttta catcatgctt atcagggtga 720ctacaagcaa ttcttggaaa gcaatgcagt caagtacctt gagggtcatg gctttcgctt 780tgaggtcagg aaaaaggaag gagtcaagcg actcgaagaa ttgcttcctg ctgcatccag 840tggcaagagc atcaggagaa cactggctgc aatgcctgaa gaggagacaa cagaagcaaa 900tgccggacag ttcctctctt ttgctagctt atttcttcct aagctagttg tcggagaaaa 960agcctgtcta gaaaaggtgc agcggcaaat tcaagttcat tctgagcagg gattgatcca 1020ataccccaca gcctggcagt cagttggaca catgatggtc attttcagac tgatgagaac 1080aaattttcta attaagttcc tccttataca tcaagggatg catatggtag caggacacga 1140tgctaacgat gctgtcatcg caaactctgt agctcaagca cgtttttcag gattattgat 1200cgttaaaaca gtgctagatc acatccttca gaaaacagag cacggagtgc gtcttcatcc 1260tttggcaaga actgctaagg tcaagaacga agtaaattcc tttaaggctg cccttagctc 1320gctagcacaa catggagagt atgctccttt tgctcgcttg ctgaatcttt ctggagtcaa 1380caatctcgag cacggactgt ttcctcagct ttctgcaatt gccctaggtg tcgcaacggc 1440acacggcagt accctggcag gagtaaatgt gggggaacag tatcagcaac tacgagaagc 1500agccactgag gcagaaaaac aattgcagaa atacgctgaa tctcgcgagc ttgaccatct 1560aggtctcgat gatcaagaga agaagatctt gaaagacttc catcagaaga aaaatgaaat 1620cagcttccag cagacaacag ccatggtcac actacggaag gaaaggctag ccaagctcac 1680tgaggcaatc acctccacat cccttctcaa gacaggaaaa cagtatgatg atgacaacga 1740tatccccttt cctgggccca tcaatgataa cgaaaactca gaacagcaag acgatgatcc 1800aacagattct caggacacta ccatccctga tatcattgtt gacccggatg atggcagata 1860caacaattat ggagactatc ctagtgagac ggcgaatgcc cctgaagacc ttgttctttt 1920tgaccttgaa gatggtgacg aggatgatca ccgaccgtca agttcatcag agaacaacaa 1980caaacacagt cttacaggaa ctgacagtaa caaaacaagt aactggaatc gaaacccgac 2040taatatgcca aagaaagact ccacacaaaa caatgacaat cctgcacagc gggctcaaga 2100atacgccagg gataacatcc aggatacacc aacaccccat cgagctctaa ctcccatcag 2160cgaagaaacc ggctccaatg gtcacaatga agatgacatt gatagcatcc ctcctttgga 2220atcagacgaa gaaaacaaca ctgagacaac cattaccacc acaaaaaata ccactgctcc 2280accagcacct gtttatcgga gtaattcaga aaaggagccc ctcccgcaag aaaaatccca 2340gaagcaacca aaccaagtga gtggtagtga gaataccgac aataaacctc actcagagca 2400atcagtggaa gaaatgtatc gacacatcct ccaaacacaa ggaccatttg atgccatcct 2460atactattac atgatgacgg aggagccgat tgtctttagc actagtgatg ggaaagaata 2520cgtataccct gattctcttg aaggggagca tccaccgtgg ctcagtgaaa aagaggcctt 2580gaatgaggac aataggttta tcacaatgga tgatcaacaa ttctactggc ctgtaatgaa 2640tcacaggaac aaattcatgg ctatccttca gcaccacaag taatttcttc ataatgacag 2700atcattgtaa ggttattacc accatccctg caacaaagca tgaaaaccac actcaacaac 2760gccctaccac aggatacctt ggagaccata caccaagatc agcagctgtg caaccacccc 2820catgcgaatc caccaccaca accaccaaac aataatccca agaccaaacc gcacacatcc 2880agatcaaccc aaaccctcaa acaccacccc actccgcgat cccagaccaa actccgcccc 2940agacaagcac cccacccatc ccagaaaccg cacggccgag aatcgatccc cagcattcaa 3000aatgcgttat taagaaaaaa catatgatga agattaaaac cttcatcaac attgcacaga 3060ctttgatcct taggagttta ttctagctat ctacaaaacg ggtccaaaac ggaatgattt 3120ccactagggc tgcagcaatc aatgatcctt cattaccaat cagaaaccag tgtacacgtg 3180gccctgaact atcaggatgg atctccgaac aattaatgac aggcaaaatt ccggtacatg 3240aaatcttcaa cgacactgag ccccacataa gctcagggtc cgactgcctt cccagaccca 3300aaaacacggc cccccggact cgcaacaccc agacacagac cgatccggtt tgcaatcaca 3360attttgaaga cgttacacaa gcactaacat cattaaccaa tgtcatacaa aaacaggctc 3420ttaacttaga gtctctcgaa caacgcatca tagatctaga gaatggctta aagccaatgt 3480atgacatggc taaagtcatt tctgcattga atagatcttg tgctgagatg gtagcaaaat 3540atgatctcct ggtgatgaca actggccgcg caaccgccac cgccgctgca actgaggctt 3600attgggagga acatggacaa ccaccacctg gaccatcact ttatgaagag agtgcgatta 3660gaggcaagat taacaagcaa gaggataaag tacctaagga agttcaagaa gcttttcgta 3720atctggacag taccagctca ctaacagaag agaactttgg caagccagat atatctgcaa 3780aggacctacg agacatcatg tatgaccacc taccaggctt cggtacggct tttcaccaac 3840tggtccaggt aatttgcaag ctaggaaaag acaattctgc attggacatt attcatgctg 3900agttccaagc cagccttgct gaaggtgatt ctccccaatg tgccctgatc caaataacaa 3960aacggatccc catcttccag gatgccactc cgcccacaat tcacatccgc tctcgtggtg 4020acatcccacg tgcctgccaa aaaagtctcc gtccagttcc tccatcacca aaaatagaca 4080gaggttgggt ttgcattttc caattgcagg acgggaagac acttgggctc aagatatagg 4140gtcccccagt caaagacacg tgcggtccca tcctccctca ccttcagaca tcaacgcatg 4200gcagtcccaa acaccggtga gggaggcgcc cggcgacaac acatgatgat aggctgatct 4260tcgggataag agacatgaaa aaccaaaaag ccgtttacat ccagatccaa gatcaagagt 4320ggcttggaaa taaggggcac ttgttctttg tctcaaagga cttacaaaaa caagggtgat 4380gaagattaag aaaaagcctc cttcagttgc aaggagctaa ttcttaaaac ttcatctaga 4440ctaaggataa atcgattcca atcacgatga ggagaatcat cctacccacg gcaccacctg 4500aatacatgga ggctgtttac ccaatgagaa caatgaattc tggtgcagac aacactgcca 4560gtggccctaa ttacacaaca actggtgtga tgacaaatga tactccctct aattcactcc 4620gaccagttgc agatgataat attgatcatc cgagccacac gcctaacagt gttgcctctg 4680catttatatt ggaagctatg gtgaatgtaa tatctggccc gaaagtgctg atgaagcaaa 4740tcccaatctg gcttcctctg ggtgtctctg accagaagac atatagcttt gattcaacca 4800ctgctgccat tatgctagca tcatatacca tcactcattt tggcaaaacc tcaaatcccc 4860ttgtgagaat caaccgactt ggtcctggca tacctgatca cccactacga ctcctaagaa 4920taggaaatca agccttccta caagagtttg tgctacctcc tgtacaactg ccacaatact 4980tcacttttga tctgacagcg ctgaagctga tcacccagcc actcccagcg gcaacctgga 5040cagatgaaac tccagctgtg tcaactggca cgctccgccc agggatctca ttccatccca 5100aattaaggcc tatcctgcta ccaggaagag ctggaaagaa gggctccaac tccgatctaa 5160catctcctga caaaatccag gctataatga atttcctaca agacctcaaa attgtaccaa 5220tcgatccaac caagaatatc atgggtattg aagtgccaga actcctggtt cacaggctga 5280ctgggaagaa gacaactacc aagaatggtc aaccaatcat tccaattctg ctaccaaagt 5340acattggtct tgatcctcta tctcaaggtg atctcacaat ggtgatcact caggactgtg 5400attcctgcca ctccccggcc agtcttcccc cagtcaatga aaaatgacca tgagactcaa 5460catcacactg ccagagcacc tcaccgcaag tctatacaac aatcaacccc ggcatctaca 5520acctgcaaaa accagcccat ctgatactcc tggcatcggg ggcaagacaa ggcagccaag 5580cagcagcccc cgagccgagc ccaaacccat tacacccgag cccaacaccc atccagcaac 5640ccacaaccgt caaacgcaca gatggacaag caaagaacat caagccagga gcaacacaga 5700ccccaagtct aagctgatca acccctcccg caatcccacc aacgccagca aaaatccccc 5760aactcgatac caaccccaag caaatcagct caaaccgtct atctctcccc gcttcactcc 5820acaccccaga ttcagcaaac gatcaacgca cttcttatgc cacagcttat attaagaaaa 5880agaacttgat gaagattaag gcaaccagtg gtgctatctt catctctttg atttgagtct 5940taagtgaata cacaggttct aatactgttc ttctgtccaa cggtataatt cagccaggcc 6000taagacagta gctaatcaca gtcatcatgg gagcgtcagg gattctgcaa ttgccccgtg 6060agcgcttcag gaaaacatct ttctttgttt gggtaataat cctattccat aaagtctttt 6120caatcccgtt gggggttgta cacaacaata ccctacaagt gagtgatatt gacaagtttg 6180tgtgccgaga caaactctct tcaactagcc aattgaagtc agtcgggttg aacttggagg 6240gcaatggagt agcaactgat gtaccaacgg caaccaaaag atggggtttt cgagctggtg 6300ttccaccaaa ggtggtaaat tgcgaagctg gagaatgggc tgagaactgt tataacctgg 6360ctataaagaa agttgatggt agtgagtgcc taccagaagc ccctgaggga gtgagggatt 6420ttccccgttg ccgctatgta cacaaagtct caggaactgg accatgccca ggaggactcg 6480cctttcacaa agaaggagcc ttcttcctgt atgaccgact cgcatcaaca atcatttatc 6540ggggtacaac ctttgccgaa ggagttattg catttctgat cttgcctaag gcgcgaaagg 6600attttttcca gtctcctcca ttgcatgagc ctgccaacat gaccacggat ccctccagtt 6660actatcacac gacaacaata aactacgtgg ttgataattt tggaaccaac accacagagt 6720ttctgttcca agtcgatcat ttgacgtatg tgcagctcga ggcaagattc acaccacaat 6780tccttgtcct cctaaatgaa accatctact ctgataaccg cagaagtaac acaacaggaa 6840aactaatctg gaaaataaat cccactgttg ataccagcat gggtgagtgg gctttctggg 6900aaaataaaaa aacttcacaa aaaccctttc aagtgaagag ttgtctttcg tacctgtacc 6960agaaacccag aaccaggtcc ttgacacgac agcgacggtc tctcctccca tctccgccca 7020caaccacgca gccgaagacc acaaagaatt ggtttcagag gattccactc cagtggttca 7080gatgcaaaac atcaagggaa aggacacaat gccaaccaca gtgacgggtg taccaacaac 7140cacaccctct ccatttccaa tcaatgctcg caacactgat cataccaaat catttatcgg 7200cctggagggg ccccaagaag accacagcac cacacagcct gccaagacca ccagccaacc 7260aaccaacagc acagaatcga cgacactaaa cccaacatca gagccctcca gtagaggcac 7320gggaccatcc agccccacgg tccccaacac cacagaaagc cacgccgaac ttggcaagac 7380aaccccaacc acactcccag aacagcacac tgccgccagt gccattccaa gagccgtgca 7440ccccgacgaa ctcagtggac ctggcttcct gacgaacaca atacgggggg ttacaaatct 7500cctgacagga tccagaagaa agcgaaggga tgtcactccc aatacacaac ccaaatgcaa 7560cccaaacctg cactattgga cagccttgga tgagggtgct gccataggtt tagcctggat 7620accatacttc gggccagcag ctgagggaat ttacactgaa ggcataatgg agaatcaaaa 7680tggattgatc tgtggattga ggcagctggc caacgaaacg acacaagctc ttcaattgtt 7740cttaagggca actactgagt tgcgtacatt ctctatacta aatcggaaag caatagactt 7800cttgctccaa agatggggag gaacatgtca cattctaggg cctgattgtt gcattgaacc 7860ccaagattgg accaaaaata tcactgataa aattgatcaa ataatccatg actttgtcga 7920taataatctt ccaaatcaga atgatggcag caactggtgg actggatgga aacaatgggt 7980tcctgctgga ataggaatca caggagtaat cattgctatt attgctttgc tgtgcatttg 8040caaattcatg ctttgaacta atatagcatc atactttcta atattccccc aatatgaatt 8100tttgttttcg attttattta atgatatatc ctctgtatac ctcactaatg tactcgagca 8160taatttccct gatagacttg attgtatttg atgattaagg acctcacaaa attcctgggg 8220attgaaaaga actggataac tcaataaatt ttatgctagg accacaaata cacttgatga 8280agattaagaa aaagataatc ttatgattat cattgatctt catctatacc ttaaatactc 8340tattcaagga gagtatgaca aaaccaagta gtattggata aacttgtcct gcattcaaat 8400ctgaagacat acggcttatc tattcactat tgtattagaa aatctaggga atatcatttg 8460aaactaatta gtgactaaaa cacacaactc aagtcggcca gaatggaagt tgttcatgaa 8520agaggtcgct ccaggatctc ccgacaaaac acaagggatg gacctagtca tttagtacgg 8580gcgagatcat cctctcgagc tagttatcga agtgaatacc atacaccaag gagtgcctcg 8640cagatccgtg tccccactgt ctttcatcgg aaaaagacag atttattgac agttccacca 8700gcacctaaag atgtatgccc gactttaaag aaagggtttc tatgtgacag caatttctgt 8760aaaaaggatc accaacttga aagcttaaca gatagagagt tactcttgct gattgcacgc 8820aagacatgtg

gatccacgga acaacaacta agcatagttg ctccaaaaga ttcacgtctg 8880gctaatccta ttgctgagga tttccaacaa aaagatgggc ctaaggtaac actgtcgatg 8940cttatagaga cagcagagta ttggtccaaa caggacatta agaacatcga tgattcaaga 9000ttaagagctt tattgaccct ttgtgctgtt atgacgcgca aattttcaaa atctcaactt 9060agcttgctat gtgaaagcca cttacggcga gaaggacttg gtcaagacca atcagagtca 9120gttctggagg tatatcaacg cttacacagc gataaaggtg ggaatttcga ggcagcacta 9180tggcagcagt gggatcggca atcattgata atgttcataa cagcattttt aaatattgca 9240ttacaattac catgtgagag ttcatctgtt gttatttcag gtttgagaat gctgataccc 9300cagtcggaag ccactgaggt tgtaaccccc tccgaaacct gcacatggtc agaaggagga 9360agttcccatt gaagccccaa atcacaaggc gagctaaaaa atcccttttg aacatgcata 9420acatcacata caatttcaaa ggcattggaa taaatggtga tttcaggaag attagtgttt 9480gccctcaaaa tcagatccga gcaataatca tctactctac agccagttaa tttctaatat 9540aaaggttaaa aaaatgctgc aggccagcta ttgttccaca ggtcccaatt cttcttgtta 9600aattgtagga gctagcacaa gtgatgcaat taaatgatac tagtatatac aatgccacca 9660acttaattct aagattttgt atatctcgga aattcaaaat taaatgctac gttattgatt 9720caattaagaa aaagacaatg gaccatcaaa attagttcaa tacctgaact aatgcactta 9780tagaaacagg agaaccagcc agacagcaga caaataacaa tgaaccacaa tatgttactg 9840ctataatgaa gttcgttaat tcaaaaacaa atgatgaaga ttaatgcaga tgtctaaagg 9900ataaacactc catgcatcag tgttataatt gggctctgta gaaaatcttc atctcctcca 9960acctacctca aagaaggatt ttaccgcgat tgggagttat aacgacaata gggacaacca 10020cctttgacac tagccaagct tgtcgtgggc acacagcatt ttatcttgca acgtcgacat 10080tcccatcaat ctgaggagta acagctatca aaacaacgca tatgtagaca ttgtcggtaa 10140tagtactgcc taagacaact atttataata acagttggaa ttcatttttt cacccaagct 10200attctcaagt taacagttga aacaggactc gacccaggac aactccggat acgtaacata 10260agaaaagaac aacccttgac ccagagtgaa caagctcata ctatcaaggc taatcctcgg 10320gcctgcctgg agtccacaat ggccaaggct actgggaggt acaaccttat ctccccaaag 10380aaagatcttg aaaaagggct ggttctgaat gacctttgca ctctctcagt ggcccagacg 10440gtccagggat ggaaggttac ctgggctggg attgaatttg atgttacaca gaaagggatg 10500gccttattgc acaggctcaa gaccagtgat tttgctccag cctggtcaat gaccaggaac 10560ttatttccac atctctttca aaacccgaac tctacaattg agtcgccact ttgggcactg 10620cgggtcatac tagcagcagg tattcaagat cagctaattg atcaatcgtt gatcgaaccc 10680ttggcaggag cgctaggctt aattgctgat tggcttctta ctactggaac aaaccacttt 10740caaatgcgca cacaacaggc taaggagcaa ctaagtctaa aaatgttgtc cctggtgcga 10800tcaaacatcc taaagttcat caaccaacta gatgcactac atgttgtgaa ttacaatgga 10860cttctcagta gcattgaaat tggcaccaaa agccatacaa ttataattac ccggacaaat 10920atgggttttt tggtagagtt gcaagagcct gacaaatcag ccatgaacac cagaaaacca 10980ggaccagtca aattctccct cctccatgaa tcaaccttga agacacttgc taaaaaacct 11040gcgacccaga tgcaagcact aatcttagaa ttcaatagtt ctctcgctat ttaactcaac 11100tcatcaaaat gctaacttgt gatccttaag ctgcacctta gacttttgat aagaatacta 11160actattgatg attgtctttg acatgaggat aagaacactg cccattagat agatggggtt 11220caccattaat acacaattac ccaatcatgt taacagcagt tagatccctc aagtatatca 11280agttcattct accctttgca ttgtcactct aattaaatca cctgatacaa ttatgttaat 11340tagctagatt ctctcatttt tagacttgtt tgctagaata attgatcatc cacttgatta 11400cacatccaac tagggtctag ttcatagatt gctaataatc tttagttcaa tactaatgac 11460aaagagatta gattagctat agcttgagga agattaagaa aaagtgtctg tggggtcttt 11520ccgtgtagaa gggcacacag ccataattct tcctctttat acaacatggc tacacaacat 11580acgcaatatc cagacgcaag gttatcatca cctatagttt tagatcagtg tgatcttgtc 11640actcgtgctt gtggattgta ttccgcatac tccttaaatc cccaactaaa gaactgtaga 11700ctaccgaaac atatataccg actaaaatat gacaccactg ttacagagtt tttgagtgat 11760gtgccggtag caacattgcc agcggatttt ttagtaccta catttcttag gactctatca 11820ggaaatggtt cttgtccaat tgatccaaaa tgcagtcaat ttttagaaga aattgtcaat 11880tatactctac aagatattcg cttcctaaac tattacctca atcgagccgg agtgcataac 11940gatcatgtgg atagggattt tggacaaaaa attcgcaatc taatttgcga caatgaggtt 12000ttacatcaaa tgtttcactg gtatgatctt gcaattctag cacgtagagg gcgactaaat 12060agagggaata atcgctcaac atggtttgca agtgataatt tggtagatat cctaggttat 12120ggagattata ttttttggaa aataccatta tcactactac cagtggatac acaaggcctc 12180ccacatgcag ccaaggactg gtatcatgaa tcggttttca aggaggctat tcaaggccat 12240acacacatcg tgtccatctc tacagcagat gtcttaatca tgtgtaagga cataatcacc 12300tgtcgattta atactttact gattgctgct gtggcaaatc tagaggattc agttcattca 12360gattaccctt taccagaaac agtgtctgac ctatacaaag caggagatta tttaatctca 12420ttgctaggat cagaaggtta caaagtcata aaattccttg agccgttatg cttagcaaag 12480atccaactct gctcaaatta cactgagagg aaaggaagat tcctcactca aatgcattta 12540gctgtaaatc atacacttga ggaacttaca gggtcccgag aattaaggcc acaacagatt 12600cggaaggtaa gggaattcca tcaaatgctg ataaacctta aggcaactcc tcaacaactc 12660tgtgagttgt tttcagtgca aaagcattgg gggcaccctg tcttgcatag cgaaaaggct 12720atccaaaaag taaagaagca tgcaacagtg ataaaagcat tgcgcccaat aataatcttt 12780gaaacatatt gtgtgtttaa atacagcatt gcaaaacatt attttgatag tcagggtacg 12840tggtacagtg tgacttctga cagatgctta acaccaggcc tttcctctta catcaaaaga 12900aaccaatttc ctccactacc tatgatcaaa gaacttttgt gggaatttta tcacttagat 12960catcctccgt tattctccac caaagtgatt agtgatttga gtatctttat taaagatcgt 13020gctactgcag tcgagaaaac atgctgggac gcagtttttg aacccaatgt tcttggttat 13080aacccaccga ataaatttgc tacaaaaagg gtacctgagc aattccttga acaggagaat 13140ttctcaatag agagtgtcct acattatgct caacgtctgg aatatcttct cccggagtac 13200cggaacttct ctttttcact caaggagaag gagttaaaca ttggacgagc ttttgggaaa 13260ttgccatatc caacacgcaa tgttcaaact ctgtgcgaag ctttgttagc agatggtttg 13320gcgaaagcat tcccaagcaa tatgatggtt gtgacagagc gcgagcaaaa agaaagcctt 13380ttgcatcaag cgtcttggca tcacacaagt gatgattttg gtgagaatgc tactgttaga 13440ggcagtagtt ttgtaacaga cttggaaaaa tacaatttag cattccgata tgagtttaca 13500gctcctttta ttgaatactg taatcgttgt tacggtgtaa gaaatttgtt taattggatg 13560cactacacta taccacagtg ttatatacat gtgagtgatt attataaccc cccacatgga 13620gtctctctcg aaaaccgaga aaatccacca gaaggtccaa gctcttaccg tggtcatcta 13680ggcgggattg agggacttca acaaaaactc tggacaagca tctcatgtgc acagatttca 13740ttagttgaaa tcaaaaccgg ttttaaactg cgatctgcgg taatgggtga caatcaatgt 13800ataactgtac tctctgtatt tcccctcgaa actgagtcta gtgagcaaga attaagttct 13860gaagataatg ccgctagagt agctgctagc ttagcaaaag tcacaagtgc ctgcggcatc 13920tttttaaaac ctgatgaaac ttttgttcac tcaggtttca tttattttgg caaaaaacaa 13980tatttgaatg gagtacaatt acctcaatca ctgaaaactg ctactagaat tgcacccttg 14040tcagatgcta tctttgatga tcttcaaggg acactagcta gcataggcac ggcttttgaa 14100agatctatct ccgaaactag gcacgtagtc ccttgtagag tagcagctgc attccatacc 14160tttttttccg taagaatctt acaatatcat catcttggct tcaacaaggg aacagacctg 14220ggtcaattgt cattaagcaa gccattagat tttggaacta taactttggc cttggcagta 14280ccacaagtct tgggtggctt atcattccta aatccagaaa aatgttttta tagaaatctg 14340ggtgatcctg ttacttcagg gctgtttcag ctcaagacat atcttcaaat gatccacatg 14400gatgatttgt ttttaccttt gatcgcaaag aacccaggga actgtagcgc aattgacttt 14460gtgttaaacc ctagtgggtt aaacgtaccg gggtcacagg atttgacatc cttcctacgt 14520cagatagtgc gccgaacaat tactctaagt gctaaaaata aattaataaa cactttgttc 14580cattcttctg ctgatttaga agatgaaatg gtttgcaaat ggttgctttc ttctacacca 14640gtcatgagta ggtttgccgc cgatatattt tctcgcactc ccagtgggaa acgtttacag 14700atcttaggtt accttgaagg gactagaaca ttgttagcct ctaaaattat aaatcataat 14760actgagacac ctatcctaga tcgattgagg aaaattacgc tgcaaaggtg gagcctgtgg 14820tttagttatc tcgaccactg tgatcaagtt ctggctgatg ccctaactca gataacctgc 14880actgtggact tagcacagat tcttcgcgag tacacctggg cacacatact agagggaagg 14940cagctcattg gagcaacact tccttgtata ctagaacaac taaatgtcat ctggctcaaa 15000ccatatgagc attgccctaa atgtgcaaag tcagcaaacc ctaaagggga accttttgtt 15060tctattgcaa ttaaaaaaca tgtagtaagt gcttggcctg atcaatcacg acttagttgg 15120acaattggag atggcatccc ttatatcgga tctcgaacag aggataagat tgggcagcca 15180gccatcaaac caaaatgccc ttcagcagcc ttacgtgaag caattgagtt gacatcaaga 15240ttgacttggg ttactcaagg tggagcaaac agcgacttac tagttaaacc cttcatagaa 15300gcacgagtaa atttaagcgt acaggaaatt ctccaaatga caccttctca ttactccggc 15360aacattgtgc atcgatataa tgatcaatat agtccacact catttatggc aaataggatg 15420agtaattctg ctactaggtt agttgtttcg acaaacactc ttggagaatt ttcaggagga 15480ggtcagtcag caagagatag taatattatc ttccagaatg tcattaattt tgctgttgca 15540ctttttgatc tacgatttag gaacgtggct acttcttcta tacaacatca tcgggctcat 15600cttcatttgt caaagtgttg cacgcgagag gttccagccc aatatttagt ttatacatca 15660acattgccat tggaccttac acggtatcgg gataatgagt tgatttacga tgacaatcca 15720ttaagaggtg gtttaaattg caatctttct tttgataatc cgcttttcaa gggccagaga 15780cttaacataa ttgaagaaga cttgattaga ctaccttact tatcaggatg ggagctagct 15840aaaactgtta tccaatctat aatttctgac agcaacaatt catcaacgga tccaatcagt 15900agtggggaaa cacgatcatt caccactcac ttcttgacat atcctaagat tggactacta 15960tatagttttg gtgcactcat cagttattat ctaggcaaca ccattattag aaccaaaaaa 16020ttgactctta acaacttcat atattaccta gctactcaaa tacataattt acctcatcgc 16080tcgttgagaa tccttaaacc tactttgaaa cacgctagtg ttatctcgag attaataagt 16140attgactctc acttctcaat ttatattgga ggaactgctg gtgatcgagg actttccgat 16200gcggcaagat tgtttcttag aactgccatt actgtcttcc ttcaattcgt tagaaagtgg 16260atagttgaac gcaagacagc tattccactg tgggtcatct accctctaga aggtcaaagt 16320cctagtccga tcaacagttt tctacaccac gtcatcgcat tgttgcaaca tgagtcctcc 16380cacgatcatg tttgtgctgc agaagcccac agtcgagtgg agacatttga taatttagtt 16440tatatgtgta aaagcacagc aagtaacttc tttcatgctt cattagcata ctggagaagt 16500cgatctaaaa atcaagacaa aagagagatg acaaagatat tatctttgac gcaaacggaa 16560aagaaaaatt cattcggcta tacagcacat ccagaaagca ctgctgttct tggttccctc 16620cagaccagcc ttgctccacc tccatctgct gacgaggcta catatgatag gaaaaacaaa 16680gttttgaaag cttccagacc tggcaagtat tcccagaata caaccaaagc cccacccaac 16740caaaccagtt gtcgcgatgt atctcccaat atcacaggca cagatgggtg cccttctgcc 16800aatgagggtt ctaacagcaa taacaataat ttagtctcgc acagaattgt actgccgttt 16860tttacattgt ctcataatta taacgaaaga ccctctatca gaaagtctga ggggacaaca 16920gagattgtaa ggcttactcg gcagctgagg gcaataccag acaccacaat atattgccgc 16980ttcacgggaa tagtttcttc aatgcactat aagctcgatg aagtcctttg ggaatttgat 17040aattttaagt ctgctataac acttgccgaa ggtgaaggtt cgggtgcatt actcttatta 17100caaaaatata aagtagaaac cttgtttttt aatacactag ccacagaaca cagcattgaa 17160gcagaaatta tttctggaat aactacacca agaatgcttc tccctattat gtctaggttc 17220catggtggac aaataaaagt cactttaaac aattctgcaa gccagattac cgatattact 17280aatccaagtt ggttggcaga ccaaaaatct aggatcccta agcaagtaga gattataacc 17340atggatgctg aaacaacaga aaacattaat cggtcaaaat tgtacgaagc agtccaacag 17400ctgattgtct cacatattga tccgaatgca ctcaaagttg tggttcttaa agttttctta 17460agtgacattg atggaatcct atggctgaat gataacctta cccctttgtt tgggctgggt 17520tacttgatca agccgatcac ctctagccca aaatctagtg agtggtacct atgtctctca 17580aaccttcttt caacttcaag acgattacct catcagagtc atactacttg catgcatgtt 17640attcaaacag cactccagct acaaattcag aggagctcat attggcttag ccaccttgtc 17700cagtatgcca atcataattt gcatttagat tatattaatc tcggtttccc ttcattggag 17760agggttttat accatagata caatttagtc gattctcaga aaggcccttt gacttccatt 17820gtccaacatc tagcgcacct gcagaccgag attagggagt tggttaatga ctataatcaa 17880caaagacaaa gtcgaaccca aacatatcat ttcattaaaa caataaaagg tcgtattaca 17940aaattggtaa atgattacct taagttcttt ctaataatac aagccttaaa gcacaattgc 18000acatggcaag aggaactaag agctcttcca gatctaatta gtgtctgcac tcgattctat 18060catactcgaa actgttcatg tgaaaaccgg ttcctagtac agactttata cttatcacgc 18120atgcaggatt cggaaatcaa actaatagat agattgaccg gccttcttag tctatgtcca 18180aatggttttt ttcggtaagg actcttgacg tacaaactcc acatagttat acaatggtac 18240caggacacta tatgtaaatt gaccctaaga aagagtaatt cgacacacag agttctcaag 18300tgaaacccct catctcagat tatctgtggt tgcaattcta atatccgatt gttaccccgt 18360gagtataact ccagattaat ataagaaaat accttttgtc ctgcaaattt atcttaaatt 18420caagtacata cgctccaaat cgtataaaat attaagaaaa agttaatctg cttgctttaa 18480ttataacttt aatattcgac aaatagttaa cggtctcatc actcaaaaat ttcattaaca 18540aaagaagtac tctgagtata ttcacatatc atatgtgatt aacatataag caacgcatga 18600tgcgccttcc tcttacttat tgtgttgtca cgcagtcgtt gtactacctc gaaaattcca 18660aacaataaat cgtgtctatc ccgcatttag tgtctttaat ttaagatctc aaatccaaaa 18720aactgggttt atgttgatgt aaatcaataa taccgaaatt gcttgatatt aaaataaagc 18780ttaaaggatt tttccttaaa cggtgatgtt aggtatatag gaaagctcga tcacgatgtc 18840ccttactcag aaaaagaaaa acggaagccc tattggccat ttaatcgtac acaaaaatat 18900ctttaccaaa ttgttttctc ttttttgtgt gtcca 1893511739PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus NP protein 11Met Glu Ser Arg Ala His Lys Ala Trp Met Thr His Thr Ala Ser Gly1 5 10 15Phe Glu Thr Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln 20 25 30Gln Gly Ile Val Arg Gln Arg Val Ile Gln Val His Gln Val Thr Asn 35 40 45Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val 50 55 60Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His65 70 75 80His Ala Tyr Gln Gly Asp Tyr Lys Gln Phe Leu Glu Ser Asn Ala Val 85 90 95Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Arg Lys Lys Glu 100 105 110Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Ala Ser Ser Gly Lys 115 120 125Ser Ile Arg Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu 130 135 140Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys145 150 155 160Leu Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile 165 170 175Gln Val His Ser Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln 180 185 190Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe 195 200 205Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly 210 215 220His Asp Ala Asn Asp Ala Val Ile Ala Asn Ser Val Ala Gln Ala Arg225 230 235 240Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln 245 250 255Lys Thr Glu His Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys 260 265 270Val Lys Asn Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala 275 280 285Gln His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly 290 295 300Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala305 310 315 320Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val 325 330 335Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys 340 345 350Gln Leu Gln Lys Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu 355 360 365Asp Asp Gln Glu Lys Lys Ile Leu Lys Asp Phe His Gln Lys Lys Asn 370 375 380Glu Ile Ser Phe Gln Gln Thr Thr Ala Met Val Thr Leu Arg Lys Glu385 390 395 400Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ser Thr Ser Leu Leu Lys 405 410 415Thr Gly Lys Gln Tyr Asp Asp Asp Asn Asp Ile Pro Phe Pro Gly Pro 420 425 430Ile Asn Asp Asn Glu Asn Ser Glu Gln Gln Asp Asp Asp Pro Thr Asp 435 440 445Ser Gln Asp Thr Thr Ile Pro Asp Ile Ile Val Asp Pro Asp Asp Gly 450 455 460Arg Tyr Asn Asn Tyr Gly Asp Tyr Pro Ser Glu Thr Ala Asn Ala Pro465 470 475 480Glu Asp Leu Val Leu Phe Asp Leu Glu Asp Gly Asp Glu Asp Asp His 485 490 495Arg Pro Ser Ser Ser Ser Glu Asn Asn Asn Lys His Ser Leu Thr Gly 500 505 510Thr Asp Ser Asn Lys Thr Ser Asn Trp Asn Arg Asn Pro Thr Asn Met 515 520 525Pro Lys Lys Asp Ser Thr Gln Asn Asn Asp Asn Pro Ala Gln Arg Ala 530 535 540Gln Glu Tyr Ala Arg Asp Asn Ile Gln Asp Thr Pro Thr Pro His Arg545 550 555 560Ala Leu Thr Pro Ile Ser Glu Glu Thr Gly Ser Asn Gly His Asn Glu 565 570 575Asp Asp Ile Asp Ser Ile Pro Pro Leu Glu Ser Asp Glu Glu Asn Asn 580 585 590Thr Glu Thr Thr Ile Thr Thr Thr Lys Asn Thr Thr Ala Pro Pro Ala 595 600 605Pro Val Tyr Arg Ser Asn Ser Glu Lys Glu Pro Leu Pro Gln Glu Lys 610 615 620Ser Gln Lys Gln Pro Asn Gln Val Ser Gly Ser Glu Asn Thr Asp Asn625 630 635 640Lys Pro His Ser Glu Gln Ser Val Glu Glu Met Tyr Arg His Ile Leu 645 650 655Gln Thr Gln Gly Pro Phe Asp Ala Ile Leu Tyr Tyr Tyr Met Met Thr 660 665 670Glu Glu Pro Ile Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Val Tyr 675 680 685Pro Asp Ser Leu Glu Gly Glu His Pro Pro Trp Leu Ser Glu Lys Glu 690 695 700Ala Leu Asn Glu Asp Asn Arg Phe Ile Thr Met Asp Asp Gln Gln Phe705 710 715 720Tyr Trp Pro Val Met Asn His Arg Asn Lys Phe Met Ala Ile Leu Gln 725 730 735His His Lys12341PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus VP35 NP protein 12Met Ile Ser Thr Arg Ala Ala Ala Ile Asn Asp Pro Ser Leu Pro Ile1 5 10 15Arg Asn Gln Cys Thr Arg Gly Pro Glu Leu Ser Gly Trp Ile Ser Glu 20 25 30Gln Leu Met Thr Gly Lys Ile Pro Val His Glu Ile Phe Asn Asp Thr 35 40

45Glu Pro His Ile Ser Ser Gly Ser Asp Cys Leu Pro Arg Pro Lys Asn 50 55 60Thr Ala Pro Arg Thr Arg Asn Thr Gln Thr Gln Thr Asp Pro Val Cys65 70 75 80Asn His Asn Phe Glu Asp Val Thr Gln Ala Leu Thr Ser Leu Thr Asn 85 90 95Val Ile Gln Lys Gln Ala Leu Asn Leu Glu Ser Leu Glu Gln Arg Ile 100 105 110Ile Asp Leu Glu Asn Gly Leu Lys Pro Met Tyr Asp Met Ala Lys Val 115 120 125Ile Ser Ala Leu Asn Arg Ser Cys Ala Glu Met Val Ala Lys Tyr Asp 130 135 140Leu Leu Val Met Thr Thr Gly Arg Ala Thr Ala Thr Ala Ala Ala Thr145 150 155 160Glu Ala Tyr Trp Glu Glu His Gly Gln Pro Pro Pro Gly Pro Ser Leu 165 170 175Tyr Glu Glu Ser Ala Ile Arg Gly Lys Ile Asn Lys Gln Glu Asp Lys 180 185 190Val Pro Lys Glu Val Gln Glu Ala Phe Arg Asn Leu Asp Ser Thr Ser 195 200 205Ser Leu Thr Glu Glu Asn Phe Gly Lys Pro Asp Ile Ser Ala Lys Asp 210 215 220Leu Arg Asp Ile Met Tyr Asp His Leu Pro Gly Phe Gly Thr Ala Phe225 230 235 240His Gln Leu Val Gln Val Ile Cys Lys Leu Gly Lys Asp Asn Ser Ala 245 250 255Leu Asp Ile Ile His Ala Glu Phe Gln Ala Ser Leu Ala Glu Gly Asp 260 265 270Ser Pro Gln Cys Ala Leu Ile Gln Ile Thr Lys Arg Ile Pro Ile Phe 275 280 285Gln Asp Ala Thr Pro Pro Thr Ile His Ile Arg Ser Arg Gly Asp Ile 290 295 300Pro Arg Ala Cys Gln Lys Ser Leu Arg Pro Val Pro Pro Ser Pro Lys305 310 315 320Ile Asp Arg Gly Trp Val Cys Ile Phe Gln Leu Gln Asp Gly Lys Thr 325 330 335Leu Gly Leu Lys Ile 34013326PRTBundibugyo ebolavirusmisc_featureCote d'Ivoire ebolavirus VP40 NP protein 13Met Arg Arg Ile Ile Leu Pro Thr Ala Pro Pro Glu Tyr Met Glu Ala1 5 10 15Val Tyr Pro Met Arg Thr Met Asn Ser Gly Ala Asp Asn Thr Ala Ser 20 25 30Gly Pro Asn Tyr Thr Thr Thr Gly Val Met Thr Asn Asp Thr Pro Ser 35 40 45Asn Ser Leu Arg Pro Val Ala Asp Asp Asn Ile Asp His Pro Ser His 50 55 60Thr Pro Asn Ser Val Ala Ser Ala Phe Ile Leu Glu Ala Met Val Asn65 70 75 80Val Ile Ser Gly Pro Lys Val Leu Met Lys Gln Ile Pro Ile Trp Leu 85 90 95Pro Leu Gly Val Ser Asp Gln Lys Thr Tyr Ser Phe Asp Ser Thr Thr 100 105 110Ala Ala Ile Met Leu Ala Ser Tyr Thr Ile Thr His Phe Gly Lys Thr 115 120 125Ser Asn Pro Leu Val Arg Ile Asn Arg Leu Gly Pro Gly Ile Pro Asp 130 135 140His Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu Gln Glu145 150 155 160Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe Asp Leu 165 170 175Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr Trp Thr 180 185 190Asp Glu Thr Pro Ala Val Ser Thr Gly Thr Leu Arg Pro Gly Ile Ser 195 200 205Phe His Pro Lys Leu Arg Pro Ile Leu Leu Pro Gly Arg Ala Gly Lys 210 215 220Lys Gly Ser Asn Ser Asp Leu Thr Ser Pro Asp Lys Ile Gln Ala Ile225 230 235 240Met Asn Phe Leu Gln Asp Leu Lys Ile Val Pro Ile Asp Pro Thr Lys 245 250 255Asn Ile Met Gly Ile Glu Val Pro Glu Leu Leu Val His Arg Leu Thr 260 265 270Gly Lys Lys Thr Thr Thr Lys Asn Gly Gln Pro Ile Ile Pro Ile Leu 275 280 285Leu Pro Lys Tyr Ile Gly Leu Asp Pro Leu Ser Gln Gly Asp Leu Thr 290 295 300Met Val Ile Thr Gln Asp Cys Asp Ser Cys His Ser Pro Ala Ser Leu305 310 315 320Pro Pro Val Asn Glu Lys 32514676PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus GP NP protein 14Met Gly Ala Ser Gly Ile Leu Gln Leu Pro Arg Glu Arg Phe Arg Lys1 5 10 15Thr Ser Phe Phe Val Trp Val Ile Ile Leu Phe His Lys Val Phe Ser 20 25 30Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 35 40 45Asp Lys Phe Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65 70 75 80Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 85 90 95Val Asn Cys Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Ala 100 105 110Ile Lys Lys Val Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 115 120 125Val Arg Asp Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Pro Gly Gly Leu Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Gly Thr Thr Phe 165 170 175Ala Glu Gly Val Ile Ala Phe Leu Ile Leu Pro Lys Ala Arg Lys Asp 180 185 190Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 195 200 205Pro Ser Ser Tyr Tyr His Thr Thr Thr Ile Asn Tyr Val Val Asp Asn 210 215 220Phe Gly Thr Asn Thr Thr Glu Phe Leu Phe Gln Val Asp His Leu Thr225 230 235 240Tyr Val Gln Leu Glu Ala Arg Phe Thr Pro Gln Phe Leu Val Leu Leu 245 250 255Asn Glu Thr Ile Tyr Ser Asp Asn Arg Arg Ser Asn Thr Thr Gly Lys 260 265 270Leu Ile Trp Lys Ile Asn Pro Thr Val Asp Thr Ser Met Gly Glu Trp 275 280 285Ala Phe Trp Glu Asn Lys Lys Asn Phe Thr Lys Thr Leu Ser Ser Glu 290 295 300Glu Leu Ser Phe Val Pro Val Pro Glu Thr Gln Asn Gln Val Leu Asp305 310 315 320Thr Thr Ala Thr Val Ser Pro Pro Ile Ser Ala His Asn His Ala Ala 325 330 335Glu Asp His Lys Glu Leu Val Ser Glu Asp Ser Thr Pro Val Val Gln 340 345 350Met Gln Asn Ile Lys Gly Lys Asp Thr Met Pro Thr Thr Val Thr Gly 355 360 365Val Pro Thr Thr Thr Pro Ser Pro Phe Pro Ile Asn Ala Arg Asn Thr 370 375 380Asp His Thr Lys Ser Phe Ile Gly Leu Glu Gly Pro Gln Glu Asp His385 390 395 400Ser Thr Thr Gln Pro Ala Lys Thr Thr Ser Gln Pro Thr Asn Ser Thr 405 410 415Glu Ser Thr Thr Leu Asn Pro Thr Ser Glu Pro Ser Ser Arg Gly Thr 420 425 430Gly Pro Ser Ser Pro Thr Val Pro Asn Thr Thr Glu Ser His Ala Glu 435 440 445Leu Gly Lys Thr Thr Pro Thr Thr Leu Pro Glu Gln His Thr Ala Ala 450 455 460Ser Ala Ile Pro Arg Ala Val His Pro Asp Glu Leu Ser Gly Pro Gly465 470 475 480Phe Leu Thr Asn Thr Ile Arg Gly Val Thr Asn Leu Leu Thr Gly Ser 485 490 495Arg Arg Lys Arg Arg Asp Val Thr Pro Asn Thr Gln Pro Lys Cys Asn 500 505 510Pro Asn Leu His Tyr Trp Thr Ala Leu Asp Glu Gly Ala Ala Ile Gly 515 520 525Leu Ala Trp Ile Pro Tyr Phe Gly Pro Ala Ala Glu Gly Ile Tyr Thr 530 535 540Glu Gly Ile Met Glu Asn Gln Asn Gly Leu Ile Cys Gly Leu Arg Gln545 550 555 560Leu Ala Asn Glu Thr Thr Gln Ala Leu Gln Leu Phe Leu Arg Ala Thr 565 570 575Thr Glu Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys Ala Ile Asp Phe 580 585 590Leu Leu Gln Arg Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp Cys 595 600 605Cys Ile Glu Pro Gln Asp Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp 610 615 620Gln Ile Ile His Asp Phe Val Asp Asn Asn Leu Pro Asn Gln Asn Asp625 630 635 640Gly Ser Asn Trp Trp Thr Gly Trp Lys Gln Trp Val Pro Ala Gly Ile 645 650 655Gly Ile Thr Gly Val Ile Ile Ala Ile Ile Ala Leu Leu Cys Ile Cys 660 665 670Lys Phe Met Leu 67515365PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus SGP NP protein 15Met Gly Ala Ser Gly Ile Leu Gln Leu Pro Arg Glu Arg Phe Arg Lys1 5 10 15Thr Ser Phe Phe Val Trp Val Ile Ile Leu Phe His Lys Val Phe Ser 20 25 30Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 35 40 45Asp Lys Phe Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65 70 75 80Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 85 90 95Val Asn Cys Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Ala 100 105 110Ile Lys Lys Val Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 115 120 125Val Arg Asp Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Pro Gly Gly Leu Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Gly Thr Thr Phe 165 170 175Ala Glu Gly Val Ile Ala Phe Leu Ile Leu Pro Lys Ala Arg Lys Asp 180 185 190Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 195 200 205Pro Ser Ser Tyr Tyr His Thr Thr Thr Ile Asn Tyr Val Val Asp Asn 210 215 220Phe Gly Thr Asn Thr Thr Glu Phe Leu Phe Gln Val Asp His Leu Thr225 230 235 240Tyr Val Gln Leu Glu Ala Arg Phe Thr Pro Gln Phe Leu Val Leu Leu 245 250 255Asn Glu Thr Ile Tyr Ser Asp Asn Arg Arg Ser Asn Thr Thr Gly Lys 260 265 270Leu Ile Trp Lys Ile Asn Pro Thr Val Asp Thr Ser Met Gly Glu Trp 275 280 285Ala Phe Trp Glu Asn Lys Lys Thr Ser Gln Lys Pro Phe Gln Val Lys 290 295 300Ser Cys Leu Ser Tyr Leu Tyr Gln Lys Pro Arg Thr Arg Ser Leu Thr305 310 315 320Arg Gln Arg Arg Ser Leu Leu Pro Ser Pro Pro Thr Thr Thr Gln Pro 325 330 335Lys Thr Thr Lys Asn Trp Phe Gln Arg Ile Pro Leu Gln Trp Phe Arg 340 345 350Cys Lys Thr Ser Arg Glu Arg Thr Gln Cys Gln Pro Gln 355 360 36516302PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus SSGP NP protein 16Met Gly Ala Ser Gly Ile Leu Gln Leu Pro Arg Glu Arg Phe Arg Lys1 5 10 15Thr Ser Phe Phe Val Trp Val Ile Ile Leu Phe His Lys Val Phe Ser 20 25 30Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 35 40 45Asp Lys Phe Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65 70 75 80Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 85 90 95Val Asn Cys Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Ala 100 105 110Ile Lys Lys Val Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 115 120 125Val Arg Asp Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Pro Gly Gly Leu Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Gly Thr Thr Phe 165 170 175Ala Glu Gly Val Ile Ala Phe Leu Ile Leu Pro Lys Ala Arg Lys Asp 180 185 190Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 195 200 205Pro Ser Ser Tyr Tyr His Thr Thr Thr Ile Asn Tyr Val Val Asp Asn 210 215 220Phe Gly Thr Asn Thr Thr Glu Phe Leu Phe Gln Val Asp His Leu Thr225 230 235 240Tyr Val Gln Leu Glu Ala Arg Phe Thr Pro Gln Phe Leu Val Leu Leu 245 250 255Asn Glu Thr Ile Tyr Ser Asp Asn Arg Arg Ser Asn Thr Thr Gly Lys 260 265 270Leu Ile Trp Lys Ile Asn Pro Thr Val Asp Thr Ser Met Gly Glu Trp 275 280 285Ala Phe Trp Glu Asn Lys Lys Leu His Lys Asn Pro Phe Lys 290 295 30017289PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus VP30 NP protein 17Met Glu Val Val His Glu Arg Gly Arg Ser Arg Ile Ser Arg Gln Asn1 5 10 15Thr Arg Asp Gly Pro Ser His Leu Val Arg Ala Arg Ser Ser Ser Arg 20 25 30Ala Ser Tyr Arg Ser Glu Tyr His Thr Pro Arg Ser Ala Ser Gln Ile 35 40 45Arg Val Pro Thr Val Phe His Arg Lys Lys Thr Asp Leu Leu Thr Val 50 55 60Pro Pro Ala Pro Lys Asp Val Cys Pro Thr Leu Lys Lys Gly Phe Leu65 70 75 80Cys Asp Ser Asn Phe Cys Lys Lys Asp His Gln Leu Glu Ser Leu Thr 85 90 95Asp Arg Glu Leu Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly Ser Thr 100 105 110Glu Gln Gln Leu Ser Ile Val Ala Pro Lys Asp Ser Arg Leu Ala Asn 115 120 125Pro Ile Ala Glu Asp Phe Gln Gln Lys Asp Gly Pro Lys Val Thr Leu 130 135 140Ser Met Leu Ile Glu Thr Ala Glu Tyr Trp Ser Lys Gln Asp Ile Lys145 150 155 160Asn Ile Asp Asp Ser Arg Leu Arg Ala Leu Leu Thr Leu Cys Ala Val 165 170 175Met Thr Arg Lys Phe Ser Lys Ser Gln Leu Ser Leu Leu Cys Glu Ser 180 185 190His Leu Arg Arg Glu Gly Leu Gly Gln Asp Gln Ser Glu Ser Val Leu 195 200 205Glu Val Tyr Gln Arg Leu His Ser Asp Lys Gly Gly Asn Phe Glu Ala 210 215 220Ala Leu Trp Gln Gln Trp Asp Arg Gln Ser Leu Ile Met Phe Ile Thr225 230 235 240Ala Phe Leu Asn Ile Ala Leu Gln Leu Pro Cys Glu Ser Ser Ser Val 245 250 255Val Ile Ser Gly Leu Arg Met Leu Ile Pro Gln Ser Glu Ala Thr Glu 260 265 270Val Val Thr Pro Ser Glu Thr Cys Thr Trp Ser Glu Gly Gly Ser Ser 275 280 285His 18251PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus VP24 NP protein 18Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu Ile Ser Pro Lys Lys Asp1 5 10 15Leu Glu Lys Gly Leu Val Leu Asn Asp Leu Cys Thr Leu Ser Val Ala 20 25 30Gln Thr Val Gln Gly Trp Lys Val Thr Trp Ala Gly Ile Glu Phe Asp 35 40 45Val Thr Gln Lys Gly Met Ala Leu Leu His Arg Leu Lys Thr Ser Asp 50 55 60Phe Ala Pro Ala Trp Ser Met Thr Arg Asn Leu Phe Pro His Leu Phe65 70 75 80Gln Asn Pro Asn Ser Thr Ile Glu Ser Pro Leu Trp Ala Leu Arg Val 85 90 95Ile Leu Ala Ala Gly Ile Gln Asp Gln Leu Ile Asp Gln Ser Leu Ile 100 105 110Glu Pro Leu Ala Gly Ala Leu Gly Leu Ile Ala Asp Trp Leu Leu Thr 115 120 125Thr Gly Thr Asn His Phe Gln Met Arg Thr Gln Gln Ala Lys Glu Gln 130 135 140Leu Ser Leu Lys Met Leu Ser Leu Val Arg Ser Asn Ile Leu Lys Phe145

150 155 160Ile Asn Gln Leu Asp Ala Leu His Val Val Asn Tyr Asn Gly Leu Leu 165 170 175Ser Ser Ile Glu Ile Gly Thr Lys Ser His Thr Ile Ile Ile Thr Arg 180 185 190Thr Asn Met Gly Phe Leu Val Glu Leu Gln Glu Pro Asp Lys Ser Ala 195 200 205Met Asn Thr Arg Lys Pro Gly Pro Val Lys Phe Ser Leu Leu His Glu 210 215 220Ser Thr Leu Lys Thr Leu Ala Lys Lys Pro Ala Thr Gln Met Gln Ala225 230 235 240Leu Ile Leu Glu Phe Asn Ser Ser Leu Ala Ile 245 250192210PRTBundibugyo ebolavirusmisc_featureCote dIvoire ebolavirus L NP protein 19Met Ala Thr Gln His Thr Gln Tyr Pro Asp Ala Arg Leu Ser Ser Pro1 5 10 15Ile Val Leu Asp Gln Cys Asp Leu Val Thr Arg Ala Cys Gly Leu Tyr 20 25 30Ser Ala Tyr Ser Leu Asn Pro Gln Leu Lys Asn Cys Arg Leu Pro Lys 35 40 45His Ile Tyr Arg Leu Lys Tyr Asp Thr Thr Val Thr Glu Phe Leu Ser 50 55 60Asp Val Pro Val Ala Thr Leu Pro Ala Asp Phe Leu Val Pro Thr Phe65 70 75 80Leu Arg Thr Leu Ser Gly Asn Gly Ser Cys Pro Ile Asp Pro Lys Cys 85 90 95Ser Gln Phe Leu Glu Glu Ile Val Asn Tyr Thr Leu Gln Asp Ile Arg 100 105 110Phe Leu Asn Tyr Tyr Leu Asn Arg Ala Gly Val His Asn Asp His Val 115 120 125Asp Arg Asp Phe Gly Gln Lys Ile Arg Asn Leu Ile Cys Asp Asn Glu 130 135 140Val Leu His Gln Met Phe His Trp Tyr Asp Leu Ala Ile Leu Ala Arg145 150 155 160Arg Gly Arg Leu Asn Arg Gly Asn Asn Arg Ser Thr Trp Phe Ala Ser 165 170 175Asp Asn Leu Val Asp Ile Leu Gly Tyr Gly Asp Tyr Ile Phe Trp Lys 180 185 190Ile Pro Leu Ser Leu Leu Pro Val Asp Thr Gln Gly Leu Pro His Ala 195 200 205Ala Lys Asp Trp Tyr His Glu Ser Val Phe Lys Glu Ala Ile Gln Gly 210 215 220His Thr His Ile Val Ser Ile Ser Thr Ala Asp Val Leu Ile Met Cys225 230 235 240Lys Asp Ile Ile Thr Cys Arg Phe Asn Thr Leu Leu Ile Ala Ala Val 245 250 255Ala Asn Leu Glu Asp Ser Val His Ser Asp Tyr Pro Leu Pro Glu Thr 260 265 270Val Ser Asp Leu Tyr Lys Ala Gly Asp Tyr Leu Ile Ser Leu Leu Gly 275 280 285Ser Glu Gly Tyr Lys Val Ile Lys Phe Leu Glu Pro Leu Cys Leu Ala 290 295 300Lys Ile Gln Leu Cys Ser Asn Tyr Thr Glu Arg Lys Gly Arg Phe Leu305 310 315 320Thr Gln Met His Leu Ala Val Asn His Thr Leu Glu Glu Leu Thr Gly 325 330 335Ser Arg Glu Leu Arg Pro Gln Gln Ile Arg Lys Val Arg Glu Phe His 340 345 350Gln Met Leu Ile Asn Leu Lys Ala Thr Pro Gln Gln Leu Cys Glu Leu 355 360 365Phe Ser Val Gln Lys His Trp Gly His Pro Val Leu His Ser Glu Lys 370 375 380Ala Ile Gln Lys Val Lys Lys His Ala Thr Val Ile Lys Ala Leu Arg385 390 395 400Pro Ile Ile Ile Phe Glu Thr Tyr Cys Val Phe Lys Tyr Ser Ile Ala 405 410 415Lys His Tyr Phe Asp Ser Gln Gly Thr Trp Tyr Ser Val Thr Ser Asp 420 425 430Arg Cys Leu Thr Pro Gly Leu Ser Ser Tyr Ile Lys Arg Asn Gln Phe 435 440 445Pro Pro Leu Pro Met Ile Lys Glu Leu Leu Trp Glu Phe Tyr His Leu 450 455 460Asp His Pro Pro Leu Phe Ser Thr Lys Val Ile Ser Asp Leu Ser Ile465 470 475 480Phe Ile Lys Asp Arg Ala Thr Ala Val Glu Lys Thr Cys Trp Asp Ala 485 490 495Val Phe Glu Pro Asn Val Leu Gly Tyr Asn Pro Pro Asn Lys Phe Ala 500 505 510Thr Lys Arg Val Pro Glu Gln Phe Leu Glu Gln Glu Asn Phe Ser Ile 515 520 525Glu Ser Val Leu His Tyr Ala Gln Arg Leu Glu Tyr Leu Leu Pro Glu 530 535 540Tyr Arg Asn Phe Ser Phe Ser Leu Lys Glu Lys Glu Leu Asn Ile Gly545 550 555 560Arg Ala Phe Gly Lys Leu Pro Tyr Pro Thr Arg Asn Val Gln Thr Leu 565 570 575Cys Glu Ala Leu Leu Ala Asp Gly Leu Ala Lys Ala Phe Pro Ser Asn 580 585 590Met Met Val Val Thr Glu Arg Glu Gln Lys Glu Ser Leu Leu His Gln 595 600 605Ala Ser Trp His His Thr Ser Asp Asp Phe Gly Glu Asn Ala Thr Val 610 615 620Arg Gly Ser Ser Phe Val Thr Asp Leu Glu Lys Tyr Asn Leu Ala Phe625 630 635 640Arg Tyr Glu Phe Thr Ala Pro Phe Ile Glu Tyr Cys Asn Arg Cys Tyr 645 650 655Gly Val Arg Asn Leu Phe Asn Trp Met His Tyr Thr Ile Pro Gln Cys 660 665 670Tyr Ile His Val Ser Asp Tyr Tyr Asn Pro Pro His Gly Val Ser Leu 675 680 685Glu Asn Arg Glu Asn Pro Pro Glu Gly Pro Ser Ser Tyr Arg Gly His 690 695 700Leu Gly Gly Ile Glu Gly Leu Gln Gln Lys Leu Trp Thr Ser Ile Ser705 710 715 720Cys Ala Gln Ile Ser Leu Val Glu Ile Lys Thr Gly Phe Lys Leu Arg 725 730 735Ser Ala Val Met Gly Asp Asn Gln Cys Ile Thr Val Leu Ser Val Phe 740 745 750Pro Leu Glu Thr Glu Ser Ser Glu Gln Glu Leu Ser Ser Glu Asp Asn 755 760 765Ala Ala Arg Val Ala Ala Ser Leu Ala Lys Val Thr Ser Ala Cys Gly 770 775 780Ile Phe Leu Lys Pro Asp Glu Thr Phe Val His Ser Gly Phe Ile Tyr785 790 795 800Phe Gly Lys Lys Gln Tyr Leu Asn Gly Val Gln Leu Pro Gln Ser Leu 805 810 815Lys Thr Ala Thr Arg Ile Ala Pro Leu Ser Asp Ala Ile Phe Asp Asp 820 825 830Leu Gln Gly Thr Leu Ala Ser Ile Gly Thr Ala Phe Glu Arg Ser Ile 835 840 845Ser Glu Thr Arg His Val Val Pro Cys Arg Val Ala Ala Ala Phe His 850 855 860Thr Phe Phe Ser Val Arg Ile Leu Gln Tyr His His Leu Gly Phe Asn865 870 875 880Lys Gly Thr Asp Leu Gly Gln Leu Ser Leu Ser Lys Pro Leu Asp Phe 885 890 895Gly Thr Ile Thr Leu Ala Leu Ala Val Pro Gln Val Leu Gly Gly Leu 900 905 910Ser Phe Leu Asn Pro Glu Lys Cys Phe Tyr Arg Asn Leu Gly Asp Pro 915 920 925Val Thr Ser Gly Leu Phe Gln Leu Lys Thr Tyr Leu Gln Met Ile His 930 935 940Met Asp Asp Leu Phe Leu Pro Leu Ile Ala Lys Asn Pro Gly Asn Cys945 950 955 960Ser Ala Ile Asp Phe Val Leu Asn Pro Ser Gly Leu Asn Val Pro Gly 965 970 975Ser Gln Asp Leu Thr Ser Phe Leu Arg Gln Ile Val Arg Arg Thr Ile 980 985 990Thr Leu Ser Ala Lys Asn Lys Leu Ile Asn Thr Leu Phe His Ser Ser 995 1000 1005Ala Asp Leu Glu Asp Glu Met Val Cys Lys Trp Leu Leu Ser Ser 1010 1015 1020Thr Pro Val Met Ser Arg Phe Ala Ala Asp Ile Phe Ser Arg Thr 1025 1030 1035Pro Ser Gly Lys Arg Leu Gln Ile Leu Gly Tyr Leu Glu Gly Thr 1040 1045 1050Arg Thr Leu Leu Ala Ser Lys Ile Ile Asn His Asn Thr Glu Thr 1055 1060 1065Pro Ile Leu Asp Arg Leu Arg Lys Ile Thr Leu Gln Arg Trp Ser 1070 1075 1080Leu Trp Phe Ser Tyr Leu Asp His Cys Asp Gln Val Leu Ala Asp 1085 1090 1095Ala Leu Thr Gln Ile Thr Cys Thr Val Asp Leu Ala Gln Ile Leu 1100 1105 1110Arg Glu Tyr Thr Trp Ala His Ile Leu Glu Gly Arg Gln Leu Ile 1115 1120 1125Gly Ala Thr Leu Pro Cys Ile Leu Glu Gln Leu Asn Val Ile Trp 1130 1135 1140Leu Lys Pro Tyr Glu His Cys Pro Lys Cys Ala Lys Ser Ala Asn 1145 1150 1155Pro Lys Gly Glu Pro Phe Val Ser Ile Ala Ile Lys Lys His Val 1160 1165 1170Val Ser Ala Trp Pro Asp Gln Ser Arg Leu Ser Trp Thr Ile Gly 1175 1180 1185Asp Gly Ile Pro Tyr Ile Gly Ser Arg Thr Glu Asp Lys Ile Gly 1190 1195 1200Gln Pro Ala Ile Lys Pro Lys Cys Pro Ser Ala Ala Leu Arg Glu 1205 1210 1215Ala Ile Glu Leu Thr Ser Arg Leu Thr Trp Val Thr Gln Gly Gly 1220 1225 1230Ala Asn Ser Asp Leu Leu Val Lys Pro Phe Ile Glu Ala Arg Val 1235 1240 1245Asn Leu Ser Val Gln Glu Ile Leu Gln Met Thr Pro Ser His Tyr 1250 1255 1260Ser Gly Asn Ile Val His Arg Tyr Asn Asp Gln Tyr Ser Pro His 1265 1270 1275Ser Phe Met Ala Asn Arg Met Ser Asn Ser Ala Thr Arg Leu Val 1280 1285 1290Val Ser Thr Asn Thr Leu Gly Glu Phe Ser Gly Gly Gly Gln Ser 1295 1300 1305Ala Arg Asp Ser Asn Ile Ile Phe Gln Asn Val Ile Asn Phe Ala 1310 1315 1320Val Ala Leu Phe Asp Leu Arg Phe Arg Asn Val Ala Thr Ser Ser 1325 1330 1335Ile Gln His His Arg Ala His Leu His Leu Ser Lys Cys Cys Thr 1340 1345 1350Arg Glu Val Pro Ala Gln Tyr Leu Val Tyr Thr Ser Thr Leu Pro 1355 1360 1365Leu Asp Leu Thr Arg Tyr Arg Asp Asn Glu Leu Ile Tyr Asp Asp 1370 1375 1380Asn Pro Leu Arg Gly Gly Leu Asn Cys Asn Leu Ser Phe Asp Asn 1385 1390 1395Pro Leu Phe Lys Gly Gln Arg Leu Asn Ile Ile Glu Glu Asp Leu 1400 1405 1410Ile Arg Leu Pro Tyr Leu Ser Gly Trp Glu Leu Ala Lys Thr Val 1415 1420 1425Ile Gln Ser Ile Ile Ser Asp Ser Asn Asn Ser Ser Thr Asp Pro 1430 1435 1440Ile Ser Ser Gly Glu Thr Arg Ser Phe Thr Thr His Phe Leu Thr 1445 1450 1455Tyr Pro Lys Ile Gly Leu Leu Tyr Ser Phe Gly Ala Leu Ile Ser 1460 1465 1470Tyr Tyr Leu Gly Asn Thr Ile Ile Arg Thr Lys Lys Leu Thr Leu 1475 1480 1485Asn Asn Phe Ile Tyr Tyr Leu Ala Thr Gln Ile His Asn Leu Pro 1490 1495 1500His Arg Ser Leu Arg Ile Leu Lys Pro Thr Leu Lys His Ala Ser 1505 1510 1515Val Ile Ser Arg Leu Ile Ser Ile Asp Ser His Phe Ser Ile Tyr 1520 1525 1530Ile Gly Gly Thr Ala Gly Asp Arg Gly Leu Ser Asp Ala Ala Arg 1535 1540 1545Leu Phe Leu Arg Thr Ala Ile Thr Val Phe Leu Gln Phe Val Arg 1550 1555 1560Lys Trp Ile Val Glu Arg Lys Thr Ala Ile Pro Leu Trp Val Ile 1565 1570 1575Tyr Pro Leu Glu Gly Gln Ser Pro Ser Pro Ile Asn Ser Phe Leu 1580 1585 1590His His Val Ile Ala Leu Leu Gln His Glu Ser Ser His Asp His 1595 1600 1605Val Cys Ala Ala Glu Ala His Ser Arg Val Glu Thr Phe Asp Asn 1610 1615 1620Leu Val Tyr Met Cys Lys Ser Thr Ala Ser Asn Phe Phe His Ala 1625 1630 1635Ser Leu Ala Tyr Trp Arg Ser Arg Ser Lys Asn Gln Asp Lys Arg 1640 1645 1650Glu Met Thr Lys Ile Leu Ser Leu Thr Gln Thr Glu Lys Lys Asn 1655 1660 1665Ser Phe Gly Tyr Thr Ala His Pro Glu Ser Thr Ala Val Leu Gly 1670 1675 1680Ser Leu Gln Thr Ser Leu Ala Pro Pro Pro Ser Ala Asp Glu Ala 1685 1690 1695Thr Tyr Asp Arg Lys Asn Lys Val Leu Lys Ala Ser Arg Pro Gly 1700 1705 1710Lys Tyr Ser Gln Asn Thr Thr Lys Ala Pro Pro Asn Gln Thr Ser 1715 1720 1725Cys Arg Asp Val Ser Pro Asn Ile Thr Gly Thr Asp Gly Cys Pro 1730 1735 1740Ser Ala Asn Glu Gly Ser Asn Ser Asn Asn Asn Asn Leu Val Ser 1745 1750 1755His Arg Ile Val Leu Pro Phe Phe Thr Leu Ser His Asn Tyr Asn 1760 1765 1770Glu Arg Pro Ser Ile Arg Lys Ser Glu Gly Thr Thr Glu Ile Val 1775 1780 1785Arg Leu Thr Arg Gln Leu Arg Ala Ile Pro Asp Thr Thr Ile Tyr 1790 1795 1800Cys Arg Phe Thr Gly Ile Val Ser Ser Met His Tyr Lys Leu Asp 1805 1810 1815Glu Val Leu Trp Glu Phe Asp Asn Phe Lys Ser Ala Ile Thr Leu 1820 1825 1830Ala Glu Gly Glu Gly Ser Gly Ala Leu Leu Leu Leu Gln Lys Tyr 1835 1840 1845Lys Val Glu Thr Leu Phe Phe Asn Thr Leu Ala Thr Glu His Ser 1850 1855 1860Ile Glu Ala Glu Ile Ile Ser Gly Ile Thr Thr Pro Arg Met Leu 1865 1870 1875Leu Pro Ile Met Ser Arg Phe His Gly Gly Gln Ile Lys Val Thr 1880 1885 1890Leu Asn Asn Ser Ala Ser Gln Ile Thr Asp Ile Thr Asn Pro Ser 1895 1900 1905Trp Leu Ala Asp Gln Lys Ser Arg Ile Pro Lys Gln Val Glu Ile 1910 1915 1920Ile Thr Met Asp Ala Glu Thr Thr Glu Asn Ile Asn Arg Ser Lys 1925 1930 1935Leu Tyr Glu Ala Val Gln Gln Leu Ile Val Ser His Ile Asp Pro 1940 1945 1950Asn Ala Leu Lys Val Val Val Leu Lys Val Phe Leu Ser Asp Ile 1955 1960 1965Asp Gly Ile Leu Trp Leu Asn Asp Asn Leu Thr Pro Leu Phe Gly 1970 1975 1980Leu Gly Tyr Leu Ile Lys Pro Ile Thr Ser Ser Pro Lys Ser Ser 1985 1990 1995Glu Trp Tyr Leu Cys Leu Ser Asn Leu Leu Ser Thr Ser Arg Arg 2000 2005 2010Leu Pro His Gln Ser His Thr Thr Cys Met His Val Ile Gln Thr 2015 2020 2025Ala Leu Gln Leu Gln Ile Gln Arg Ser Ser Tyr Trp Leu Ser His 2030 2035 2040Leu Val Gln Tyr Ala Asn His Asn Leu His Leu Asp Tyr Ile Asn 2045 2050 2055Leu Gly Phe Pro Ser Leu Glu Arg Val Leu Tyr His Arg Tyr Asn 2060 2065 2070Leu Val Asp Ser Gln Lys Gly Pro Leu Thr Ser Ile Val Gln His 2075 2080 2085Leu Ala His Leu Gln Thr Glu Ile Arg Glu Leu Val Asn Asp Tyr 2090 2095 2100Asn Gln Gln Arg Gln Ser Arg Thr Gln Thr Tyr His Phe Ile Lys 2105 2110 2115Thr Ile Lys Gly Arg Ile Thr Lys Leu Val Asn Asp Tyr Leu Lys 2120 2125 2130Phe Phe Leu Ile Ile Gln Ala Leu Lys His Asn Cys Thr Trp Gln 2135 2140 2145Glu Glu Leu Arg Ala Leu Pro Asp Leu Ile Ser Val Cys Thr Arg 2150 2155 2160Phe Tyr His Thr Arg Asn Cys Ser Cys Glu Asn Arg Phe Leu Val 2165 2170 2175Gln Thr Leu Tyr Leu Ser Arg Met Gln Asp Ser Glu Ile Lys Leu 2180 2185 2190Ile Asp Arg Leu Thr Gly Leu Leu Ser Leu Cys Pro Asn Gly Phe 2195 2200 2205Phe Arg 22102018959DNAZaire ebolavirusmisc_featureFull viral sequence 20cggacacaca aaaagaaaga agaattttta ggatcttttg tgtgcgaata actatgagga 60agattaataa ttttcctctc attgaaattt atatcggaat ttaaattgaa attgttactg 120taatcacacc tggtttgttt cagagccaca tcacaaagat agagaacaac ctaggtctcc 180gaagggagca agggcatcag tgtgctcagt tgaaaatccc ttgtcaacac ctaggtctta 240tcacatcaca agttccacct cagactctgc agggtgatcc aacaacctta atagaaacat 300tattgttaaa ggacagcatt agttcacagt caaacaagca agattgagaa ttaaccttgg 360ttttgaactt gaacacttag gggattgaag attcaacaac cctaaagctt ggggtaaaac 420attggaaata gttaaaagac aaattgctcg gaatcacaaa attccgagta tggattctcg 480tcctcagaaa atctggatgg cgccgagtct cactgaatct gacatggatt accacaagat 540cttgacagca ggtctgtccg ttcaacaggg gattgttcgg caaagagtca tcccagtgta 600tcaagtaaac aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt 660tgattttcaa gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca 720gggagattac aaacttttct tggaaagtgg cgcagtcaag tatttggaag

ggcacgggtt 780ccgttttgaa gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt 840atctagtgga aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga 900agctaatgcc ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg 960agaaaaggct tgccttgaga aggttcaaag gcaaattcaa gtacatgcag agcaaggact 1020gatacaatat ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat 1080gcgaacaaat tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg 1140gcatgatgcc aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt 1200attgattgtc aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct 1260ccatcctctt gcaaggaccg ccaaggtaaa aaatgaggtg aactccttta aggctgcact 1320cagctccctg gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg 1380agtaaataat cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc 1440cacagcacac gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag 1500agaggctgcc actgaggctg agaagcaact ccaacaatat gcagagtctc gcgaacttga 1560ccatcttgga cttgatgatc aggaaaagaa aattcttatg aacttccatc agaaaaagaa 1620cgaaatcagc ttccagcaaa caaacgctat ggtaactcta agaaaagagc gcctggccaa 1680gctgacagaa gctatcactg ctgcgtcact gcccaaaaca agtggacatt acgatgatga 1740tgacgacatt ccctttccag gacccatcaa tgatgacgac aatcctggcc atcaagatga 1800tgatccgact gactcacagg atacgaccat tcccgatgtg gtggttgatc ccgatgatgg 1860aagctacggc gaataccaga gttactcgga aaacggcatg aatgcaccag atgacttggt 1920cctattcgat ctagacgagg acgacgagga cactaagcca gtgcctaata gatcgaccaa 1980gggtggacaa cagaagaaca gtcaaaaggg ccagcatata gagggcagac agacacaatc 2040caggccaatt caaaatgtcc caggccctca cagaacaatc caccacgcca gtgcgccact 2100cacggacaat gacagaagaa atgaaccctc cggctcaacc agccctcgca tgctgacacc 2160aattaacgaa gaggcagacc cactggacga tgccgacgac gagacgtcta gccttccgcc 2220cttggagtca gatgatgaag agcaggacag ggacggaact tccaaccgca cacccactgt 2280cgccccaccg gctcccgtat acagagatca ctctgaaaag aaagaactcc cgcaagacga 2340gcaacaagat caggaccaca ctcaagaggc caggaaccag gacagtgaca acacccagtc 2400agaacactct tttgaggaga tgtatcgcca cattctaaga tcacaggggc catttgatgc 2460tgttttgtat tatcatatga tgaaggatga gcctgtagtt ttcagtacca gtgatggcaa 2520agagtacacg tatccagact cccttgaaga ggaatatcca ccatggctca ctgaaaaaga 2580ggctatgaat gaagagaata gatttgttac attggatggt caacaatttt attggccggt 2640gatgaatcac aagaataaat tcatggcaat cctgcaacat catcagtgaa tgagcatgga 2700acaatgggat gattcaaccg acaaatagct aacattaagt agtcaaggaa cgaaaacagg 2760aagaattttt gatgtctaag gtgtgaatta ttatcacaat aaaagtgatt cttatttttg 2820aatttaaagc tagcttatta ttactagccg tttttcaaag ttcaatttga gtcttaatgc 2880aaataggcgt taagccacag ttatagccat aattgtaact caatattcta actagcgatt 2940tatctaaatt aaattacatt atgcttttat aacttaccta ctagcctgcc caacatttac 3000acgatcgttt tataattaag aaaaaactaa tgatgaagat taaaaccttc atcatcctta 3060cgtcaattga attctctagc actcgaagct tattgtcttc aatgtaaaag aaaagctggt 3120ctaacaagat gacaactaga acaaagggca ggggccatac tgcggccacg actcaaaacg 3180acagaatgcc aggccctgag ctttcgggct ggatctctga gcagctaatg accggaagaa 3240ttcctgtaag cgacatcttc tgtgatattg agaacaatcc aggattatgc tacgcatccc 3300aaatgcaaca aacgaagcca aacccgaaga cgcgcaacag tcaaacccaa acggacccaa 3360tttgcaatca tagttttgag gaggtagtac aaacattggc ttcattggct actgttgtgc 3420aacaacaaac catcgcatca gaatcattag aacaacgcat tacgagtctt gagaatggtc 3480taaagccagt ttatgatatg gcaaaaacaa tctcctcatt gaacagggtt tgtgctgaga 3540tggttgcaaa atatgatctt ctggtgatga caaccggtcg ggcaacagca accgctgcgg 3600caactgaggc ttattgggcc gaacatggtc aaccaccacc tggaccatca ctttatgaag 3660aaagtgcgat tcggggtaag attgaatcta gagatgagac cgtccctcaa agtgttaggg 3720aggcattcaa caatctaaac agtaccactt cactaactga ggaaaatttt gggaaacctg 3780acatttcggc aaaggatttg agaaacatta tgtatgatca cttgcctggt tttggaactg 3840ctttccacca attagtacaa gtgatttgta aattgggaaa agatagcaac tcattggaca 3900tcattcatgc tgagttccag gccagcctgg ctgaaggaga ctctcctcaa tgtgccctaa 3960ttcaaattac aaaaagagtt ccaatcttcc aagatgctgc tccacctgtc atccacatcc 4020gctctcgagg tgacattccc cgagcttgcc agaaaagctt gcgtccagtc ccaccatcgc 4080ccaagattga tcgaggttgg gtatgtgttt ttcagcttca agatggtaaa acacttggac 4140tcaaaatttg agccaatctc ccttccctcc gaaagaggcg aataatagca gaggcttcaa 4200ctgctgaact atagggtacg ttacattaat gatacacttg tgagtatcag ccctggataa 4260tataagtcaa ttaaacgacc aagataaaat tgttcatatc tcgctagcag cttaaaatat 4320aaatgtaata ggagctatat ctctgacagt attataatca attgttatta agtaacccaa 4380accaaaagtg atgaagatta agaaaaacct acctcggctg agagagtgtt ttttcattaa 4440ccttcatctt gtaaacgttg agcaaaattg ttaaaaatat gaggcgggtt atattgccta 4500ctgctcctcc tgaatatatg gaggccatat accctgtcag gtcaaattca acaattgcta 4560gaggtggcaa cagcaataca ggcttcctga caccggagtc agtcaatggg gacactccat 4620cgaatccact caggccaatt gccgatgaca ccatcgacca tgccagccac acaccaggca 4680gtgtgtcatc agcattcatc cttgaagcta tggtgaatgt catatcgggc cccaaagtgc 4740taatgaagca aattccaatt tggcttcctc taggtgtcgc tgatcaaaag acctacagct 4800ttgactcaac tacggccgcc atcatgcttg cttcatacac tatcacccat ttcggcaagg 4860caaccaatcc acttgtcaga gtcaatcggc tgggtcctgg aatcccggat catcccctca 4920ggctcctgcg aattggaaac caggctttcc tccaggagtt cgttcttccg ccagtccaac 4980taccccagta tttcaccttt gatttgacag cactcaaact gatcacccaa ccactgcctg 5040ctgcaacatg gaccgatgac actccaacag gatcaaatgg agcgttgcgt ccaggaattt 5100catttcatcc aaaacttcgc cccattcttt tacccaacaa aagtgggaag aaggggaaca 5160gtgccgatct aacatctccg gagaaaatcc aagcaataat gacttcactc caggacttta 5220agatcgttcc aattgatcca accaaaaata tcatgggaat cgaagtgcca gaaactctgg 5280tccacaagct gaccggtaag aaggtgactt ctaaaaatgg acaaccaatc atccctgttc 5340ttttgccaaa gtacattggg ttggacccgg tggctccagg agacctcacc atggtaatca 5400cacaggattg tgacacgtgt cattctcctg caagtcttcc agctgtgatt gagaagtaat 5460tgcaataatt gactcagatc cagttttata gaatcttctc agggatagtg ataacatcta 5520tttagtaatc cgtccattag aggagacact tttaattgat caatatacta aaggtgcttt 5580acaccattgt cttttttctc tcctaaatgt agaacttaac aaaagactca taatatactt 5640gtttttaaag gattgattga tgaaagatca taactaataa cattacaaat aatcctacta 5700taatcaatac ggtgattcaa atgttaatct ttctcattgc acatactttt tgcccttatc 5760ctcaaattgc ctgcatgctt acatctgagg atagccagtg tgacttggat tggaaatgtg 5820gagaaaaaat cgggacccat ttctaggttg ttcacaatcc aagtacagac attgcccttc 5880taattaagaa aaaatcggcg atgaagatta agccgacagt gagcgtaatc ttcatctctc 5940ttagattatt tgttttccag agtaggggtc gtcaggtcct tttcaatcgt gtaaccaaaa 6000taaactccac tagaaggata ttgtggggca acaacacaat gggcgttaca ggaatattgc 6060agttacctcg tgatcgattc aagaggacat cattctttct ttgggtaatt atccttttcc 6120aaagaacatt ttccatccca cttggagtca tccacaatag cacattacag gttagtgatg 6180tcgacaaact agtttgtcgt gacaaactgt catccacaaa tcaattgaga tcagttggac 6240tgaatctcga agggaatgga gtggcaactg acgtgccatc tgcaactaaa agatggggct 6300tcaggtccgg tgtcccacca aaggtggtca attatgaagc tggtgaatgg gctgaaaact 6360gctacaatct tgaaatcaaa aaacctgacg ggagtgagtg tctaccagca gcgccagacg 6420ggattcgggg cttcccccgg tgccggtatg tgcacaaagt atcaggaacg ggaccgtgtg 6480ccggagactt tgccttccat aaagagggtg ctttcttcct gtatgatcga cttgcttcca 6540cagttatcta ccgaggaacg actttcgctg aaggtgtcgt tgcatttctg atactgcccc 6600aagctaagaa ggacttcttc agctcacacc ccttgagaga gccggtcaat gcaacggagg 6660acccgtctag tggctactat tctaccacaa ttagatatca ggctaccggt tttggaacca 6720atgagacaga gtacttgttc gaggttgaca atttgaccta cgtccaactt gaatcaagat 6780tcacaccaca gtttctgctc cagctgaatg agacaatata tacaagtggg aaaaggagca 6840ataccacggg aaaactaatt tggaaggtca accccgaaat tgatacaaca atcggggagt 6900gggccttctg ggaaactaaa aaaacctcac tagaaaaatt cgcagtgaag agttgtcttt 6960cacagttgta tcaaacggag ccaaaaacat cagtggtcag agtccggcgc gaacttcttc 7020cgacccaggg accaacacaa caactgaaga ccacaaaatc atggcttcag aaaattcctc 7080tgcaatggtt caagtgcaca gtcaaggaag ggaagctgca gtgtcgcatc taacaaccct 7140tgccacaatc tccacgagtc cccaatccct cacaaccaaa ccaggtccgg acaacagcac 7200ccataataca cccgtgtata aacttgacat ctctgaggca actcaagttg aacaacatca 7260ccgcagaaca gacaacgaca gcacagcctc cgacactccc tctgccacga ccgcagccgg 7320acccccaaaa gcagagaaca ccaacacgag caagagcact gacttcctgg accccgccac 7380cacaacaagt ccccaaaacc acagcgagac cgctggcaac aacaacactc atcaccaaga 7440taccggagaa gagagtgcca gcagcgggaa gctaggctta attaccaata ctattgctgg 7500agtcgcagga ctgatcacag gcgggagaag aactcgaaga gaagcaattg tcaatgctca 7560acccaaatgc aaccctaatt tacattactg gactactcag gatgaaggtg ctgcaatcgg 7620actggcctgg ataccatatt tcgggccagc agccgaggga atttacatag aggggctaat 7680gcacaatcaa gatggtttaa tctgtgggtt gagacagctg gccaacgaga cgactcaagc 7740tcttcaactg ttcctgagag ccacaactga gctacgcacc ttttcaatcc tcaaccgtaa 7800ggcaattgat ttcttgctgc agcgatgggg cggcacatgc cacattctgg gaccggactg 7860ctgtatcgaa ccacatgatt ggaccaagaa cataacagac aaaattgatc agattattca 7920tgattttgtt gataaaaccc ttccggacca gggggacaat gacaattggt ggacaggatg 7980gagacaatgg ataccggcag gtattggagt tacaggcgtt ataattgcag ttatcgcttt 8040attctgtata tgcaaatttg tcttttagtt tttcttcaga ttgcttcatg gaaaagctca 8100gcctcaaatc aatgaaacca ggatttaatt atatggatta cttgaatcta agattacttg 8160acaaatgata atataataca ctggagcttt aaacatagcc aatgtgattc taactccttt 8220aaactcacag ttaatcataa acaaggtttg acatcaatct agttatctct ttgagaatga 8280taaacttgat gaagattaag aaaaaggtaa tctttcgatt atctttaatc ttcatccttg 8340attctacaat catgacagtt gtctttagtg acaagggaaa gaagcctttt tattaagttg 8400taataatcag atctgcgaac cggtagagtt tagttgcaac ctaacacaca taaagcattg 8460gtcaaaaagt caatagaaat ttaaacagtg agtggagaca acttttaaat ggaagcttca 8520tatgagagag gacgcccacg agctgccaga cagcattcaa gggatggaca cgaccaccat 8580gttcgagcac gatcatcatc cagagagaat tatcgaggtg agtaccgtca atcaaggagc 8640gcctcacaag tgcgcgttcc tactgtattt cataagaaga gagttgaacc attaacagtt 8700cctccagcac ctaaagacat atgtccgacc ttgaaaaaag gatttttgtg tgacagtagt 8760ttttgcaaaa aagatcacca gttggagagt ttaactgata gggaattact cctactaatc 8820gcccgtaaga cttgtggatc agtagaacaa caattaaata taactgcacc caaggactcg 8880cgcttagcaa atccaacggc tgatgatttc cagcaagagg aaggtccaaa aattaccttg 8940ttgacactga tcaagacggc agaacactgg gcgagacaag acatcagaac catagaggat 9000tcaaaattaa gagcattgtt gactctatgt gctgtgatga cgaggaaatt ctcaaaatcc 9060cagctgagtc ttttatgtga gacacaccta aggcgcgagg ggcttgggca agatcaggca 9120gaacccgttc tcgaagtata tcaacgatta cacagtgata aaggaggcag ttttgaagct 9180gcactatggc aacaatggga ccgacaatcc ctaattatgt ttatcactgc attcttgaat 9240attgctctcc agttaccgtg tgaaagttct gctgtcgttg tttcagggtt aagaacattg 9300gttcctcaat cagataatga ggaagcttca accaacccgg ggacatgctc atggtctgat 9360gagggtaccc cttaataagg ctgactaaaa cactatataa ccttctactt gatcacaata 9420ctccgtatac ctatcatcat atatttaatc aagacgatat cctttaaaac ttattcagta 9480ctataatcac tctcgtttca aattaataag atgtgcatga ttgccctaat atatgaagag 9540gtatgataca accctaacag tgatcaaaga aaatcataat ctcgtatcgc tcgtaatata 9600acctgccaag catacctctt gcacaaagtg attcttgtac acaaataatg ttttactcta 9660caggaggtag caacgatcca tcccatcaaa aaataagtat ttcatgactt actaatgatc 9720tcttaaaata ttaagaaaaa ctgacggaac ataaattctt tatgcttcaa gctgtggagg 9780aggtgtttgg tattggctat tgttatatta caatcaataa caagcttgta aaaatattgt 9840tcttgtttca agaggtagat tgtgaccgga aatgctaaac taatgatgaa gattaatgcg 9900gaggtctgat aagaataaac cttattattc agattaggcc ccaagaggca ttcttcatct 9960ccttttagca aagtactatt tcagggtagt ccaattagtg gcacgtcttt tagctgtata 10020tcagtcgccc ctgagatacg ccacaaaagt gtctctaagc taaattggtc tgtacacatc 10080ccatacattg tattaggggc aataatatct aattgaactt agccgtttaa aatttagtgc 10140ataaatctgg gctaacacca ccaggtcaac tccattggct gaaaagaagc ttacctacaa 10200cgaacatcac tttgagcgcc ctcacaatta aaaaatagga acgtcgttcc aacaatcgag 10260cgcaaggttt caaggttgaa ctgagagtgt ctagacaaca aaatattgat actccagaca 10320ccaagcaaga cctgagaaaa aaccatggct aaagctacgg gacgatacaa tctaatatcg 10380cccaaaaagg acctggagaa aggggttgtc ttaagcgacc tctgtaactt cttagttagc 10440caaactattc aggggtggaa ggtttattgg gctggtattg agtttgatgt gactcacaaa 10500ggaatggccc tattgcatag actgaaaact aatgactttg cccctgcatg gtcaatgaca 10560aggaatctct ttcctcattt atttcaaaat ccgaattcca caattgaatc accgctgtgg 10620gcattgagag tcatccttgc agcagggata caggaccagc tgattgacca gtctttgatt 10680gaacccttag caggagccct tggtctgatc tctgattggc tgctaacaac caacactaac 10740catttcaaca tgcgaacaca acgtgtcaag gaacaattga gcctaaaaat gctgtcgttg 10800attcgatcca atattctcaa gtttattaac aaattggatg ctctacatgt cgtgaactac 10860aacggattgt tgagcagtat tgaaattgga actcaaaatc atacaatcat cataactcga 10920actaacatgg gttttctggt ggagctccaa gaacccgaca aatcggcaat gaaccgcatg 10980aagcctgggc cggcgaaatt ttccctcctt catgagtcca cactgaaagc atttacacaa 11040ggatcctcga cacgaatgca aagtttgatt cttgaattta atagctctct tgctatctaa 11100ctaaggtaga atacttcata ttgagctaac tcatatatgc tgactcaata gttatcttga 11160catctctgct ttcataatca gatatataag cataataaat aaatactcat atttcttgat 11220aatttgttta accacagata aatcctcact gtaagccagc ttccaagttg acacccttac 11280aaaaaccagg actcagaatc cctcaaacaa gagattccaa gacaacatca tagaattgct 11340ttattatatg aataagcatt ttatcaccag aaatcctata tactaaatgg ttaattgtaa 11400ctgaacccgc aggtcacatg tgttaggttt cacagattct atatattact aactctatac 11460tcgtaattaa cattagataa gtagattaag aaaaaagcct gaggaagatt aagaaaaact 11520gcttattggg tctttccgtg ttttagatga agcagttgaa attcttcctc ttgatattaa 11580atggctacac aacataccca atacccagac gctaggttat catcaccaat tgtattggac 11640caatgtgacc tagtcactag agcttgcggg ttatattcat catactccct taatccgcaa 11700ctacgcaact gtaaactccc gaaacatatc taccgtttga aatacgatgt aactgttacc 11760aagttcttga gtgatgtacc agtggcgaca ttgcccatag atttcatagt cccagttctt 11820ctcaaggcac tgtcaggcaa tggattctgt cctgttgagc cgcggtgcca acagttctta 11880gatgaaatca ttaagtacac aatgcaagat gctctcttct tgaaatatta tctcaaaaat 11940gtgggtgctc aagaagactg tgttgatgaa cactttcaag agaaaatctt atcttcaatt 12000cagggcaatg aatttttaca tcaaatgttt ttctggtatg atctggctat tttaactcga 12060aggggtagat taaatcgagg aaactctaga tcaacatggt ttgttcatga tgatttaata 12120gacatcttag gctatgggga ctatgttttt tggaagatcc caatttcaat gttaccactg 12180aacacacaag gaatccccca tgctgctatg gactggtatc aggcatcagt attcaaagaa 12240gcggttcaag ggcatacaca cattgtttct gtttctactg ccgacgtctt gataatgtgc 12300aaagatttaa ttacatgtcg attcaacaca actctaatct caaaaatagc agagattgag 12360gatccagttt gttctgatta tcccaatttt aagattgtgt ctatgcttta ccagagcgga 12420gattacttac tctccatatt agggtctgat gggtataaaa ttattaagtt cctcgaacca 12480ttgtgcttgg ccaaaattca attatgctca aagtacactg agaggaaggg ccgattctta 12540acacaaatgc atttagctgt aaatcacacc ctagaagaaa ttacagaaat gcgtgcacta 12600aagccttcac aggctcaaaa gatccgtgaa ttccatagaa cattgataag gctggagatg 12660acgccacaac aactttgtga gctattttcc attcaaaaac actgggggca tcctgtgcta 12720catagtgaaa cagcaatcca aaaagttaaa aaacatgcta cggtgctaaa agcattacgc 12780cctatagtga ttttcgagac atactgtgtt tttaaatata gtattgccaa acattatttt 12840gatagtcaag gatcttggta cagtgttact tcagatagga atctaacacc gggtcttaat 12900tcttatatca aaagaaatca attccctccg ttgccaatga ttaaagaact actatgggaa 12960ttttaccacc ttgaccaccc tccacttttc tcaaccaaaa ttattagtga cttaagtatt 13020tttataaaag acagagctac cgcagtagaa aggacatgct gggatgcagt attcgagcct 13080aatgttctag gatataatcc acctcacaaa tttagtacta aacgtgtacc ggaacaattt 13140ttagagcaag aaaacttttc tattgagaat gttctttcct acgcacaaaa actcgagtat 13200ctactaccac aatatcggaa cttttctttc tcattgaaag agaaagagtt gaatgtaggt 13260agaaccttcg gaaaattgcc ttatccgact cgcaatgttc aaacactttg tgaagctctg 13320ttagctgatg gtcttgctaa agcatttcct agcaatatga tggtagttac ggaacgtgag 13380caaaaagaaa gcttattgca tcaagcatca tggcaccaca caagtgatga ttttggtgaa 13440catgccacag ttagagggag tagctttgta actgatttag agaaatacaa tcttgcattt 13500agatatgagt ttacagcacc ttttatagaa tattgcaacc gttgctatgg tgttaagaat 13560gtttttaatt ggatgcatta tacaatccca cagtgttata tgcatgtcag tgattattat 13620aatccaccac ataacctcac actggagaat cgagacaacc cccccgaagg gcctagttca 13680tacaggggtc atatgggagg gattgaagga ctgcaacaaa aactctggac aagtatttca 13740tgtgctcaaa tttctttagt tgaaattaag actggtttta agttacgctc agctgtgatg 13800ggtgacaatc agtgcattac tgttttatca gtcttcccct tagagactga cgcagacgag 13860caggaacaga gcgccgaaga caatgcagcg agggtggccg ccagcctagc aaaagttaca 13920agtgcctgtg gaatcttttt aaaacctgat gaaacatttg tacattcagg ttttatctat 13980tttggaaaaa aacaatattt gaatggggtc caattgcctc agtcccttaa aacggctaca 14040agaatggcac cattgtctga tgcaattttt gatgatcttc aagggaccct ggctagtata 14100ggcactgctt ttgagcgatc catctctgag acacgacata tctttccttg caggataacc 14160gcagctttcc atacgttttt ttcggtgaga atcttgcaat atcatcatct cgggttcaat 14220aaaggttttg accttggaca gttaacactc ggcaaacctc tggatttcgg aacaatatca 14280ttggcactag cggtaccgca ggtgcttgga gggttatcct tcttgaatcc tgagaaatgt 14340ttctaccgga atctaggaga tccagttacc tcaggcttat tccagttaaa aacttatctc 14400cgaatgattg agatggatga tttattctta cctttaattg cgaagaaccc tgggaactgc 14460actgccattg actttgtgct aaatcctagc ggattaaatg tccctgggtc gcaagactta 14520acttcatttc tgcgccagat tgtacgcagg accatcaccc taagtgcgaa aaacaaactt 14580attaatacct tatttcatgc gtcagctgac ttcgaagacg aaatggtttg taaatggcta 14640ttatcatcaa ctcctgttat gagtcgtttt gcggccgata tcttttcacg cacgccgagc 14700gggaagcgat tgcaaattct aggatacctg gaaggaacac gcacattatt agcctctaag 14760atcatcaaca ataatacaga gacaccggtt ttggacagac tgaggaaaat aacattgcaa 14820aggtggagcc tatggtttag ttatcttgat cattgtgata atatcctggc ggaggcttta 14880acccaaataa cttgcacagt tgatttagca cagattctga gggaatattc atgggctcat 14940attttagagg gaagacctct tattggagcc acactcccat gtatgattga gcaattcaaa 15000gtgttttggc tgaaacccta cgaacaatgt ccgcagtgtt caaatgcaaa gcaaccaggt 15060gggaaaccat tcgtgtcagt ggcagtcaag aaacatattg ttagtgcatg gccgaacgca 15120tcccgaataa gctggactat cggggatgga atcccataca ttggatcaag gacagaagat 15180aagataggac aacctgctat taaaccaaaa tgtccttccg cagccttaag agaggccatt 15240gaattggcgt cccgtttaac atgggtaact caaggcagtt cgaacagtga cttgctaata 15300aaaccatttt tggaagcacg agtaaattta agtgttcaag aaatacttca aatgacccct 15360tcacattact caggaaatat tgttcacagg tacaacgatc aatacagtcc tcattctttc 15420atggccaatc gtatgagtaa ttcagcaacg cgattgattg tttctacaaa cactttaggt 15480gagttttcag gaggtggcca gtctgcacgc gacagcaata ttattttcca gaatgttata 15540aattatgcag ttgcactgtt cgatattaaa tttagaaaca ctgaggctac agatatccaa 15600tataatcgtg ctcaccttca tctaactaag tgttgcaccc gggaagtacc agctcagtat 15660ttaacataca catctacatt ggatttagat ttaacaagat accgagaaaa cgaattgatt 15720tatgacagta atcctctaaa aggaggactc aattgcaata tctcattcga taatccattt 15780ttccaaggta aacggctgaa cattatagaa gatgatctta ttcgactgcc

tcacttatct 15840ggatgggagc tagccaagac catcatgcaa tcaattattt cagatagcaa caattcatct 15900acagacccaa ttagcagtgg agaaacaaga tcattcacta cccatttctt aacttatccc 15960aagataggac ttctgtacag ttttggggcc tttgtaagtt attatcttgg caatacaatt 16020cttcggacta agaaattaac acttgacaat tttttatatt acttaactac tcaaattcat 16080aatctaccac atcgctcatt gcgaatactt aagccaacat tcaaacatgc aagcgttatg 16140tcacggttaa tgagtattga tcctcatttt tctatttaca taggcggtgc tgcaggtgac 16200agaggactct cagatgcggc caggttattt ttgagaacgt ccatttcatc ttttcttaca 16260tttgtaaaag aatggataat taatcgcgga acaattgtcc ctttatggat agtatatccg 16320ctagagggtc aaaacccaac acctgtgaat aattttctct atcagatcgt agaactgctg 16380gtgcatgatt catcaagaca acaggctttt aaaactacca taagtgatca tgtacatcct 16440cacgacaatc ttgtttacac atgtaagagt acagccagca atttcttcca tgcatcattg 16500gcgtactgga ggagcagaca cagaaacagc aaccgaaaat acttggcaag agactcttca 16560actggatcaa gcacaaacaa cagtgatggt catattgaga gaagtcaaga acaaaccacc 16620agagatccac atgatggcac tgaacggaat ctagtcctac aaatgagcca tgaaataaaa 16680agaacgacaa ttccacaaga aaacacgcac cagggtccgt cgttccagtc ctttctaagt 16740gactctgctt gtggtacagc aaatccaaaa ctaaatttcg atcgatcgag acacaatgtg 16800aaatttcagg atcataactc ggcatccaag agggaaggtc atcaaataat ctcacaccgt 16860ctagtcctac ctttctttac attatctcaa gggacacgcc aattaacgtc atccaatgag 16920tcacaaaccc aagacgagat atcaaagtac ttacggcaat tgagatccgt cattgatacc 16980acagtttatt gtagatttac cggtatagtc tcgtccatgc attacaaact tgatgaggtc 17040ctttgggaaa tagagagttt caagtcggct gtgacgctag cagagggaga aggtgctggt 17100gccttactat tgattcagaa ataccaagtt aagaccttat ttttcaacac gctagctact 17160gagtccagta tagagtcaga aatagtatca ggaatgacta ctcctaggat gcttctacct 17220gttatgtcaa aattccataa tgaccaaatt gagattattc ttaacaactc agcaagccaa 17280ataacagaca taacaaatcc tacttggttt aaagaccaaa gagcaaggct acctaagcaa 17340gtcgaggtta taaccatgga tgcagagaca acagagaata taaacagatc gaaattgtac 17400gaagctgtat ataaattgat cttacaccat attgatccta gcgtattgaa agcagtggtc 17460cttaaagtct ttctaagtga tactgagggt atgttatggc taaatgataa tttagccccg 17520ttttttgcca ctggttattt aattaagcca ataacgtcaa gtgctagatc tagtgagtgg 17580tatctttgtc tgacgaactt cttatcaact acacgtaaga tgccacacca aaaccatctc 17640agttgtaaac aggtaatact tacggcattg caactgcaaa ttcaacgaag cccatactgg 17700ctaagtcatt taactcagta tgctgactgt gagttacatt taagttatat ccgccttggt 17760tttccatcat tagagaaagt actataccac aggtataacc tcgtcgattc aaaaagaggt 17820ccactagtct ctatcactca gcacttagca catcttagag cagagattcg agaattaact 17880aatgattata atcaacagcg acaaagtcgg actcaaacat atcactttat tcgtactgca 17940aaaggacgaa tcacaaaact agtcaatgat tatttaaaat tctttcttat tgtgcaagca 18000ttaaaacata atgggacatg gcaagctgag tttaagaaat taccagagtt gattagtgtg 18060tgcaataggt tctaccatat tagagattgc aattgtgaag aacgtttctt agttcaaacc 18120ttatatttac atagaatgca ggattctgaa gttaagctta tcgaaaggct gacagggctt 18180ctgagtttat ttccggatgg tctctacagg tttgattgaa ttaccgtgca tagtatcctg 18240atacttgcaa aggttggtta ttaacataca gattataaaa aactcataaa ttgctctcat 18300acatcatatt gatctaatct caataaacaa ctatttaaat aacgaaagga gtccctatat 18360tatatactat atttagcctc tctccctgcg tgataatcaa aaaattcaca atgcagcatg 18420tgtgacatat tactgccgca atgaatttaa cgcaacataa taaactctgc actctttata 18480attaagcttt aacgaaaggt ctgggctcat attgttattg atataataat gttgtatcaa 18540tatcctgtca gatggaatag tgttttggtt gataacacaa cttcttaaaa caaaattgat 18600ctttaagatt aagtttttta taattatcat tactttaatt tgtcgtttta aaaacggtga 18660tagccttaat ctttgtgtaa aataagagat taggtgtaat aaccttaaca tttttgtcta 18720gtaagctact atttcataca gaatgataaa attaaaagaa aaggcaggac tgtaaaatca 18780gaaatacctt ctttacaata tagcagacta gataataatc ttcgtgttaa tgataattaa 18840gacattgacc acgctcatca gaaggctcgc cagaataaac gttgcaaaaa ggattcctgg 18900aaaaatggtc gcacacaaaa atttaaaaat aaatctattt cttctttttt gtgtgtcca 189592120DNAArtificial SequencePCR forward primer for Sudan ebola BMG 21gccatggntt caggtttgag 202222DNAArtificial SequencePCR reverse primer for Sudan ebola BMG 22ggtnacattg ggcaacaatt ca 222326DNAArtificial SequencePCR probe for Sudan ebola BMG 23acggtgcaca ttctcctttt ctcgga 262422DNAArtificial SequencePCR forward primer for Ebola Bundibugyo fragment A 24gtgagacaaa gaatcattcc tg 222523DNAArtificial SequencePCR reverse primer for Ebola Bundibugyo fragment A 25catcaattgc tcagagatcc acc 232621DNAArtificial SequencePCR forward primer for Ebola Bundibugyo fragment B 26ccaacaacac tgcatgtaag t 212720DNAArtificial SequencePCR reverse primer for Ebola Bundibugyo fragment B 27aggtcgcgtt aatcttcatc 202821DNAArtificial SequencePCR forward primer for Ebola Bundibugyo fragment C 28gatggttgag ttactttccg g 212920DNAArtificial SequencePCR reverse primer for Ebola Bundibugyo fragment C 29gtcttgagtc atcaatgccc 203018DNAArtificial SequencePCR forward primer for Ebola Bundibugyo fragment D 30ccaccagcac caaaggac 183122DNAArtificial SequencePCR reverse primer for Ebola Bundibugyo fragment D 31ctatcggcaa tgtaactatt gg 223219DNAArtificial SequencePCR forward primer for Ebola Bundibugyo fragment E 32gccgttgtag aggacacac 193326DNAArtificial SequencePCR reverse primer for Ebola Bundibugyo fragment E 33cacattaaat tgttctaaca tgcaag 263423DNAArtificial SequencePCR forward primer for Ebola Bundibugyo fragment F 34cctaggttat ttagaaggga cta 233524DNAArtificial SequencePCR reverse primer for Ebola Bundibugyo fragment F 35ggtagatgta ttgacagcaa tatc 243621DNAArtificial SequencePCR primer for Ebola Uganda 692(-) 36acaaaaagct atctgcacta t 213720DNAArtificial SequencePCR primer for Ebola Uganda 18269(+) 37ctcagaagca aaattaatgg 203823DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment A 38gtgtgcgaat aactatgagg aag 233922DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment A 39gtctgtgcaa tgttgatgaa gg 224022DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment B 40catgaaaacc acactcaaca ac 224124DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment B 41gttgccttaa tcttcatcaa gttc 244222DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment C 42ggctataatg aatttcctcc ag 224322DNAArtificial SequencePCR reverse primer for ebola cote dIviore virus fragment C 43caagtgtatt tgtggtccta gc 224420DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment C 44gctggaatag gaatcacagg 204521DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment D 45cggtagtcta cagttcttta g 214625DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment E 46gacaaagaga ttagattagc tatag 254722DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment E 47gtaatgagaa ggtgtcattt gg 224822DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment F 48cacgacttag ttggacaatt gg 224923DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment F 49cagacactaa ttagatctgg aag 235021DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment G 50cggacacaca aaaagaawra a 215121DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment G 51cgttcttgac cttagcagtt c 215222DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus fragment H 52gcactataag ctcgatgaag tc 225320DNAArtificial SequencePCR reverse primer for Cote dIviore ebola virus fragment H 53tggacacaca aaaargaraa 205421DNAArtificial SequencePCR forward primer for Cote dIviore ebola virus gap between fragments C and D 54ctgagaggat ccagaagaaa g 215522DNAArtificial SequencePCR reverse primer for Cote d'Iviore ebola virus gap between fragments C and D 55gtgtaagcgt tgatatacct cc 225622DNAArtificial SequencePCR forward primer for ebola uganda virus EboU965(+) 56gagaaaaggc ctgtctggag aa 225724DNAArtificial SequencePCR forward primer for ebola uganda virus EboU1039(-) 57tcgggtattg aatcagacct tgtt 245826DNAArtificial SequencePCR probe for ebola uganda virus EboU989 58ttcaacgaca aatccaagtg cacgca 2659302PRTBundibugyo ebolavirusmisc_featureSSGP viral protein 59Met Val Thr Ser Gly Ile Leu Gln Leu Pro Arg Glu Arg Phe Arg Lys1 5 10 15Thr Ser Phe Phe Val Trp Val Ile Ile Leu Phe His Lys Val Phe Pro 20 25 30Ile Pro Leu Gly Val Val His Asn Asn Thr Leu Gln Val Ser Asp Ile 35 40 45Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Ser Gln Leu Lys 50 55 60Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp Val Pro65 70 75 80Thr Ala Thr Lys Arg Trp Gly Phe Arg Ala Gly Val Pro Pro Lys Val 85 90 95Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn Leu Asp 100 105 110Ile Lys Lys Ala Asp Gly Ser Glu Cys Leu Pro Glu Ala Pro Glu Gly 115 120 125Val Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser Gly Thr 130 135 140Gly Pro Cys Pro Glu Gly Tyr Ala Phe His Lys Glu Gly Ala Phe Phe145 150 155 160Leu Tyr Asp Arg Leu Ala Ser Thr Ile Ile Tyr Arg Ser Thr Thr Phe 165 170 175Ser Glu Gly Val Val Ala Phe Leu Ile Leu Pro Glu Thr Lys Lys Asp 180 185 190Phe Phe Gln Ser Pro Pro Leu His Glu Pro Ala Asn Met Thr Thr Asp 195 200 205Pro Ser Ser Tyr Tyr His Thr Val Thr Leu Asn Tyr Val Ala Asp Asn 210 215 220Phe Gly Thr Asn Met Thr Asn Phe Leu Phe Gln Val Asp His Leu Thr225 230 235 240Tyr Val Gln Leu Glu Pro Arg Phe Thr Pro Gln Phe Leu Val Gln Leu 245 250 255Asn Glu Thr Ile Tyr Thr Asn Gly Arg Arg Ser Asn Thr Thr Gly Thr 260 265 270Leu Ile Trp Lys Val Asn Pro Thr Val Asp Thr Gly Val Gly Glu Trp 275 280 285Ala Phe Trp Glu Asn Lys Lys Leu His Lys Asn Pro Phe Lys 290 295 300

Claims

1. An isolated hEbola virus comprising a nucleic acid molecule comprising a nucleotide sequence of: a) a nucleotide sequence set forth in SEQ ID NOS: 1 or 10; b) a nucleotide sequence hybridizing under stringent conditions to SEQ ID NOS: 1 or 10; or c) a nucleotide sequence of at least 70%-99% identity to the SEQ ID NOS: 1 or 10, with the proviso that said nucleotide sequence is not SEQ ID NO: 20.

2. An isolated hEbola virus having Centers for Disease Control Deposit Accession No. 200706291.

3. The hEbola virus of claim 1 which is killed.

4. The hEbola virus of claim 1 which is an attenuated hEbola virus.

5. The virus of claim 4 wherein at least one property of the attenuated hEbola virus is reduced from among infectivity, replication ability, protein synthesis ability, assembling ability or cytopathic effect.

6. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS: 1 or 10 or a complement thereof, or a fragment thereof wherein said fragment comprises a nucleotide sequence of between 4 and 4900 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof; with the proviso that said nucleotide sequence is not comprised by the nucleotide sequence set forth in SEQ ID NO: 20; or between 5500 and 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof.

7. The isolated nucleic acid molecule of claim 6 comprising a nucleotide sequence of between 4 and 4900 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof; with the proviso that said nucleotide sequence is not comprised by the nucleotide sequence set forth in SEQ ID NO: 20; or between 5500 and 6600 contiguous nucleotides of the nucleotide sequence of SEQ ID NOS: 1 or 10, or a complement thereof.

8. The isolated nucleic acid molecule of claim 7 comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2-9, 59, or SEQ ID NO: 11-19 or a complement thereof.

9. An isolated RNA or DNA nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS: 1 or 10 or a complement thereof.

10. An isolated polypeptide encoded by the nucleic acid molecule of claim 7.

11. The polypeptide of claim 10 comprising the amino acid of: a) an amino acid sequence set forth in any of SEQ ID NOS: 2-19, or 59; or b) an amino acid sequence that has 70%-99% homology to the amino acid sequence of (a).

12. The polypeptide of claim 10 wherein the amino acid sequence has 5 to 250 contiguous amino acid residues of the amino acid sequence of SEQ ID NOS: 5 or 18 (VP24); 5 to 280 contiguous residues of the amino acid sequence of SEQ ID NOS: 6 or 17 (VP30); 5 to 320 contiguous residues of the amino acid sequence of SEQ ID NOS: 8 or 13 (VP40); 5 to 340 contiguous residues of the amino acid sequence of SEQ ID NOS: 7 or 12 (VP35); 5 to 370 contiguous residues of the amino acid sequence of SEQ ID NOS: 4 or 15 (SGP); 5 to 370 contiguous residues of the amino acid sequence of SEQ ID NOS: 59 or 16 (SSGP); 5 to 670 contiguous residues of the amino acid sequence of SEQ ID NOS: 9 or 14 (GP); 5 to 730 contiguous residues of the amino acid sequence of SEQ ID NOS: 3 or 11 (NP); or 5 to 2200 contiguous residues of the amino acid sequence of SEQ ID NOS: 2 or 19 (L).

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. The hEbola virus of claims 3 or 4, or a protein extract therefrom, and a pharmaceutically acceptable carrier.

21. (canceled)

22. The nucleic acid molecule of claims 6 or 9, and a pharmaceutically acceptable carrier.

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

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