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CN117343911A - Preparation method and application of defective filovirus - Google Patents

Preparation method and application of defective filovirus Download PDF

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Publication number
CN117343911A
CN117343911A CN202210754314.5A CN202210754314A CN117343911A CN 117343911 A CN117343911 A CN 117343911A CN 202210754314 A CN202210754314 A CN 202210754314A CN 117343911 A CN117343911 A CN 117343911A
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ser
thr
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Inventor
左建平
石正丽
童贤崑
杨莉
杨兴娄
周宇
杨晓倩
朱峰华
谢莎
李恒
巩颖
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Shanghai Institute of Materia Medica of CAS
Wuhan Institute of Virology of CAS
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Shanghai Institute of Materia Medica of CAS
Wuhan Institute of Virology of CAS
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Priority to CN202210754314.5A priority Critical patent/CN117343911A/en
Priority to PCT/CN2023/101148 priority patent/WO2024001845A1/en
Publication of CN117343911A publication Critical patent/CN117343911A/en
Pending legal-status Critical Current

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Abstract

The invention provides a preparation method and application of a defective type filovirus. Specifically, the invention provides a defective recombinant filovirus, wherein the coding sequence of one or more key viral proteins in the genome of the defective recombinant filovirus is replaced by a recombinase coding sequence, and the defective recombinant filovirus is used in various aspects such as antiviral drug research and development, virology research and vaccine development. The defective recombinant filovirus has good biological safety characteristics, is easy to produce, can completely simulate the related immune response of host cells caused by virus infection, and is a tool virus with extremely high practicability.

Description

Preparation method and application of defective filovirus
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of a defective type filovirus.
Background
The Filoviridae family (school name: filoviridae), the order mononegavirales, is a class of vertebrate-infecting viruses, including ebola, marburg, quinirus and yunnan. Viral particles (Virion) have complex structures with an envelope, a nucleocapsid (nucleoapsid), a polymerase complex and a Matrix protein (Matrix). The outer layer of the virus particle has a capsule structure. The virus is in a filiform shape, or has a branched polymorphic shape, or is U-shaped, 6-shaped or circular (especially after purification), the diameter of the virus is about 80nm, the virus can reach 14000nm long, and the length of the purified virus can reach 790-970nm. The surface has the shape of tumor-like protrusions dispersed in the lipid bilayer membrane.
Ebola virus (EBOV) in the family of filoviridae is a deadly virus that, after infection, can cause hemorrhagic fever disease in humans and non-human primates. Ebola Virus has been determined to contain 6 species, namely zaire Virus (Ebola Virus), sudan Virus (Sudan Virus), leston Virus (Reston Virus), bendi Jiao Bingdu (Bundibugyo Virus), tarift forest VirusForest virus) and bunbary virus (bonbali virus). Different species have different characteristics, the most dangerous of which is zaire-type ebola disease, with mortality rates of up to 90%, in epidemic areas mortality rates of 88% in 1976, 100% in 1977, 59% in 1994, 81% in 1995, 73% in 1996, 80% in 2001 to 2002, 90% in 2003, and an average of 83% in 2007.
Currently, there is no antiviral drug approved for use against ebola virus, which makes it necessary to conduct ebola virus-related research work in biosafety class 4 (BSL-4) laboratories, limited to the limited resources of high-level biosafety laboratories, and difficult progress in ebola virus-related viral biology research and therapeutic drug development.
Therefore, in order to bypass this obstacle, there is an urgent need in the art to develop a novel ebola virus research model that can be used to develop studies of viral biology and antiviral treatment techniques in low-level biosafety laboratories.
Disclosure of Invention
The invention aims to provide a preparation method and application of a defective recombinant filovirus, which are widely used in various aspects such as antiviral drug research and development, virology research and vaccine development.
In a first aspect of the invention there is provided a defective recombinant filovirus, the genome of which has been replaced with recombinase coding sequences for one or more key viral proteins,
wherein the key viral protein is selected from the group consisting of: GP protein, VP40 protein, VP24 protein, or a combination thereof; the recombinase is selected from the group consisting of: cre recombinase, flp recombinase, dre recombinase, or a combination thereof.
In another preferred embodiment, the defective recombinant filovirus includes, but is not limited to: ebola virus, marburg virus.
In another preferred embodiment, the defective recombinant filovirus has a similarity to the wild-type filovirus of greater than or equal to 90%, preferably greater than or equal to 95%.
In another preferred embodiment, the antigenic characteristics of the defective recombinant filovirus are identical to those of a wild-type filovirus.
In another preferred embodiment, the genome structure of the defective recombinant filovirus from 3 'end to 5' end is as shown in formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8 (I)
In the method, in the process of the invention,
each "-" is independently a bond or a nucleotide linking sequence;
z1 is the coding sequence of the NP protein of the filovirus;
z2 is the coding sequence of the VP35 protein of the filovirus;
z3 is the coding sequence VP40 of the filovirus VP40 protein or the coding sequence of the recombinase;
z4 is the coding sequence GP of the filovirus GP protein or the coding sequence of the recombinase;
z5 is the coding sequence of the VP30 protein of the filovirus;
z6 is the coding sequence VP24 of the filovirus VP24 protein or the coding sequence of the recombinase;
z7 is the coding sequence of filovirus L protein;
z8 is a null or transcription assistance sequence;
and at least one of Z3, Z4 and Z6 is a recombinant enzyme coding sequence.
In another preferred embodiment, the recombinase coding sequence is selected from the group consisting of: cre recombinase coding sequence Cre, flp recombinase coding sequence Flp, dre recombinase coding sequence Dre, or a combination thereof.
In another preferred embodiment, the recombinase coding sequence is the Cre recombinase coding sequence Cre.
In another preferred embodiment, the transcription assistance sequence is selected from the group consisting of: HDV ribozyme sequence, D-glucosamine 6-phosphate riboswitch ribozyme (glucosamine-6-phosphate riboswitch ribozyme) sequence (glmS ribozyme sequence), hammerhead ribozyme (Hammerhead ribozyme) sequence, hairpin ribozyme sequence, varkud Satellite (VS) ribozyme sequence, or a combination thereof.
In another preferred embodiment, the Z8 is an HDV ribozyme sequence.
In another preferred embodiment, the genomic structure is: Z1-Z2-VP40-Cre-Z5-VP24-Z7-Z8.
In another preferred embodiment, the genomic structure is: Z1-Z2-Cre-GP-Z5-VP24-Z7-Z8.
In another preferred embodiment, the genomic structure is: Z1-Z2-VP40-GP-Z5-Cre-Z7-Z8.
In a second aspect of the invention there is provided an isolated polynucleotide encoding a defective recombinant filovirus according to the first aspect of the invention.
In another preferred embodiment, the polynucleotide may be DNA, RNA or cDNA.
In another preferred embodiment, the polynucleotide is selected from the group consisting of:
(a) A polynucleotide with a sequence shown as SEQ ID NO. 1;
(b) A polynucleotide having a nucleotide sequence having a homology of 95% (preferably 98%) or more with the sequence shown in SEQ ID NO. 1;
(c) A polynucleotide truncated at the 5 'and/or 3' end of the polynucleotide shown in SEQ ID NO. 1 or added with 1 to 99 (preferably 1 to 49, more preferably 1 to 9) nucleotides; and
(d) A polynucleotide complementary to the polynucleotide of any one of (a) - (c); and
in another preferred embodiment, the polynucleotide has a structure as shown in formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8 (I)
in the method, in the process of the invention,
Each "-" is independently a bond or a nucleotide linking sequence;
z1 to Z8 are as described above;
and at least one of Z3, Z4 and Z6 is a recombinant enzyme coding sequence.
In another preferred embodiment, the recombinase coding sequence is a sequence shown in SEQ ID NO. 5.
In another preferred embodiment, Z1 is a sequence as set forth in SEQ ID NO. 2.
In another preferred embodiment, Z2 is a sequence as set forth in SEQ ID NO. 3.
In another preferred embodiment, Z3 is a sequence as set forth in SEQ ID NO. 4.
In another preferred embodiment, Z4 is a sequence as set forth in SEQ ID NO. 5.
In another preferred embodiment, Z5 is a sequence as set forth in SEQ ID NO. 6.
In another preferred embodiment, Z6 is a sequence as set forth in SEQ ID NO. 7.
In another preferred embodiment, Z7 is a sequence as set forth in SEQ ID NO. 8.
In a third aspect of the invention there is provided a vector comprising a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, viral vectors, plasmid vectors, transposons, other gene transfer systems, or combinations thereof. Preferably, the vector is a plasmid.
In another preferred embodiment, the viral vector includes, but is not limited to: lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.
In another preferred embodiment, the plasmid vector includes, but is not limited to: bacterial artificial chromosome (BAC vector), yeast Artificial Chromosome (YAC) vector, cosmid (plasmid) vector, or a combination thereof.
In another preferred embodiment, the vector is a bacterial artificial chromosome vector.
In another preferred embodiment, the carrier comprises: a promoter element, a terminator element, or a combination thereof.
In another preferred embodiment, the promoter element includes, but is not limited to: a human polymerase I promoter, a mouse polymerase I promoter, or a combination thereof.
In another preferred embodiment, the promoter element is a human polymerase I promoter.
In another preferred embodiment, the terminator element includes, but is not limited to: a mouse polymerase I terminator, a human polymerase I terminator, or a combination thereof.
In another preferred embodiment, the terminator element is a human polymerase I terminator.
In a fourth aspect of the invention there is provided a host cell comprising a vector according to the third aspect of the invention, or having incorporated into its genome a polynucleotide according to the second aspect of the invention.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells.
In another preferred embodiment, the host cell is a HEK293 cell.
In a fifth aspect of the present invention, there is provided a composition comprising:
(a) The vector according to the third aspect of the present invention, wherein the coding sequence for the key viral protein in the vector is replaced with a recombinase coding sequence; and
(b) A regulated expression vector, wherein said regulated expression vector is regulated by expression of said recombinase of (a), and expresses said critical viral protein of (a) under suitable conditions.
In another preferred embodiment, the composition further comprises:
(c) Other viral protein expression vectors.
In another preferred embodiment, the regulatory expression vector in (b) is a pDIO-GP plasmid.
In another preferred embodiment, the regulatory expression vector has a structure as shown in formula II:
X1-X2-X3-X4-X5-X6-X7-X8 (II)
in the method, in the process of the invention,
each "-" is independently a bond or a nucleotide linking sequence;
x1 is none or a promoter;
x2 and X7 are specific recognition sites of recombinant enzyme;
x3 is none or a resistance gene;
x4 is the coding sequence of a key viral protein;
x5 is a cleavage peptide;
X6 is a reporter gene;
x8 is a poly a signal peptide;
and X2 is opposite to X7.
In another preferred embodiment, X1 is a CMV promoter.
In another preferred embodiment, the specific recognition site is selected from the group consisting of: a lox site recognized by Cre recombinase, an FRT site recognized by Flp recombinase, a rox site recognized by Dre recombinase, or a combination thereof.
In another preferred embodiment, the lox site is selected from the group consisting of: loxP site, loxN site, lox2272 site, lox5171 site, or a combination thereof.
In another preferred embodiment, the X2 is a loxP site and a loxN site.
In another preferred embodiment, the X7 is loxP site and loxN site.
In another preferred embodiment, the X3 is located intermediate two specific recognition sites.
In another preferred embodiment, the X3 includes, but is not limited to: bleomycin (Zeocin) resistance gene, neomycin (Neomycin) resistance gene, blasticidin (Blastidin S) resistance gene, puromycin (Puromycin) resistance gene, hygromycin B (HygromycinB) resistance gene, or a combination thereof.
In another preferred embodiment, the X3 is a bleomycin (Zeocin) resistance gene.
In another preferred embodiment, the X4 is selected from the group consisting of: the coding sequence for GP protein, the coding sequence for VP40 protein, the coding sequence for VP24 protein, or a combination thereof.
In another preferred embodiment, the X4 is the coding sequence of GP protein.
In another preferred embodiment, X5 is P2A or T2A.
In another preferred embodiment, the X6 includes, but is not limited to: a fluorescent protein gene, a luciferase (luciferase) gene, a luciferase-like protein (nanoLuciferase) encoding gene, a lacZ gene, a secreted alkaline phosphatase (SEAP) gene, a chloramphenicol acetyl transferase (Chloramphenicol acetyltransferase) gene, or a combination thereof.
In another preferred embodiment, the fluorescent protein genes include, but are not limited to: GFP gene, RFP gene, BFP gene, YFP gene, CFP gene.
In another preferred embodiment, the X6 is a firefly luciferase and green fluorescent protein (FLuc: GFP) fusion protein gene.
In another preferred embodiment, the other viral protein expression vector is selected from the group consisting of: NP protein expression vector, VP35 protein expression vector, VP30 protein expression vector, L protein expression vector, or a combination thereof.
In another preferred embodiment, the other viral protein expression vector has a structure as shown in formula III:
Y1-Y2-Y3 (II)
in the method, in the process of the invention,
each "-" is independently a bond or a nucleotide linking sequence;
y1 is none or a promoter;
y2 is selected from the group consisting of: the coding sequence of NP protein, the coding sequence of VP35 protein, the coding sequence of VP30 protein, the coding sequence of L protein, or a combination thereof.
Y3 is a poly A signal peptide.
In a sixth aspect of the present invention, there is provided a method for preparing a defective recombinant filovirus, the method comprising the steps of:
(a) Providing a vector according to the third aspect of the invention, wherein the coding sequence for a key viral protein in the vector is replaced with a recombinase coding sequence;
(b) Constructing a regulatory expression vector, wherein said regulatory expression vector is regulated by expression of said recombinase of (a), and expressing said critical viral protein of (a) under appropriate conditions;
(c) Transduction of the regulated expression vector of step (b) into a eukaryotic cell, thereby obtaining a complementing cell;
(d) Optionally, constructing other viral protein expression vectors; and
(e) Transduction of the vector of step (a) and optionally other viral protein expression vectors into the complementary cells obtained in step (c), thereby obtaining the defective recombinant filovirus.
In another preferred embodiment, the eukaryotic cell described in step (c) is a HEK293 cell.
In another preferred embodiment, the transduction of step (c) and step (e) uses a liposome transfection reagent.
In another preferred embodiment, the eukaryotic cells are cultured for a further 48 hours after the transduction in step (c) to obtain the complementing cells.
In another preferred embodiment, the complementing cell in step (c) is HEK293-loxGP.
In another preferred embodiment, said complementing cells are cultured for 96 hours after said transduction in step (e) to collect said defective recombinant filovirus.
In another preferred embodiment, the preparation method further comprises:
(f) Isolating and/or purifying the defective recombinant filovirus obtained in step (e); and/or
(g) Amplifying the defective recombinant filovirus obtained in step (e) or (f).
In another preferred embodiment, step (f) is performed using a 0.45 μm filter.
In another preferred embodiment, said expansion culture in step (g) is performed in said complementing cells.
In a seventh aspect of the invention there is provided the use of a composition according to the fifth aspect of the invention for the preparation of a defective recombinant filovirus according to the first aspect of the invention.
In an eighth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) Inactivated or attenuated defective recombinant filoviruses as described in the first aspect of the invention; and
(ii) A pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the pharmaceutical composition comprises a vaccine composition.
In another preferred embodiment, the vaccine composition further comprises an adjuvant.
In another preferred embodiment, the vaccine comprises 0.01 to 99.99% of the defective recombinant filovirus according to the first aspect of the invention and 0.01 to 99.99% of a pharmaceutically acceptable carrier, diluent or excipient, said percentages being mass percentages of the pharmaceutical composition.
In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injection and freeze-dried preparation.
In another preferred embodiment, the pharmaceutical composition further comprises:
(iii) Other drugs and pharmaceutically acceptable carriers, diluents or excipients thereof.
In a ninth aspect of the invention there is provided a detection reagent comprising a defective recombinant filovirus according to the first aspect of the invention, coupled with a detectable label.
In a tenth aspect of the invention, there is provided a method of detecting filovirus antibodies in a sample in vitro, the method comprising the steps of:
(1) Contacting said sample in vitro with a defective recombinant filovirus according to the first aspect of the invention; and
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of the filovirus antibody in the sample.
In another preferred embodiment, the detection comprises diagnostic or non-diagnostic.
In an eleventh aspect of the present invention, there is provided a method for producing a filovirus antibody, the method comprising the steps of: the non-human mammal is immunized with a defective recombinant filovirus according to the first aspect of the invention as an immunogen.
In another preferred embodiment, the preparation method further comprises the steps of: the defective recombinant filoviruses are mixed with a suitable adjuvant and emulsified.
In another preferred embodiment, the immunization may comprise multiple immunizations.
In another preferred embodiment, the immunization regimen includes, but is not limited to: intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, intraperitoneal injection, and the like.
In another preferred embodiment, the non-human mammal includes, but is not limited to: mice, rats, rabbits, alpacas, and the like.
In a twelfth aspect of the invention there is provided the use of a defective recombinant filovirus according to the first aspect of the invention for a standard reference substance in a method of detection of filviruses or in a kit.
In a thirteenth aspect of the invention, there is provided a kit for detection of a filovirus, the kit comprising:
(i) The defective recombinant filovirus according to the first aspect of the invention as a standard reference substance;
(ii) A filovirus antibody; and
(iii) A specification describing a method for qualitatively and quantitatively detecting filoviruses in a sample.
In another preferred embodiment, the kit further comprises an enzyme-labeled secondary antibody to the filovirus antibody.
In another preferred embodiment, the kit further comprises a color developing agent and a terminator.
In another preferred embodiment, the kit further comprises a sample diluent.
In a fourteenth aspect of the invention, there is provided a method of in vitro detection of a filovirus, the method comprising the steps of:
(i) Providing a sample to be tested, and contacting the sample to be tested, a standard reference substance and a blank with a filovirus antibody, wherein the standard reference substance is the defective recombinant filovirus according to the first aspect of the invention;
(ii) Adding an enzyme-labeled secondary antibody of the filovirus antibody, reacting with a substrate to develop color, and stopping the reaction; and
(iii) Measuring the detection value A2 of the sample to be detected, the detection value A1 of the standard reference substance and the detection value A0 of the blank control,
if A1 is more than or equal to 2A0, judging that the detection result is valid;
If A1 is less than 2A0, judging that the detection result is invalid;
if A2 is more than or equal to 2A1, judging that the sample to be detected is strong positive, and indicating that the sample to be detected contains a large amount of filoviruses;
if 0.5A1 is less than A2 and less than 2A1, judging that the sample to be detected is weak positive, and indicating that the sample to be detected contains a small amount of the filovirus;
and if A2 is less than or equal to 0.5A1, judging that the sample to be tested is negative, and indicating that the sample to be tested does not contain or basically does not contain the filovirus.
In another preferred embodiment, the filovirus antibody in step (i) is coated on an elisa plate.
In another preferred embodiment, the determination in step (iii) is performed by means of a microplate reader.
In another preferred embodiment, the method further comprises the steps of:
(iv) Drawing a standard curve by using detection values of samples to be detected with different dilutions, and quantitatively judging the content of the filovirus in the samples to be detected.
In a fifteenth aspect of the present invention, there is provided a method of anti-filovirus drug discovery and evaluation, the method comprising the steps of:
(a) Providing a drug to be tested and a defective recombinant filovirus of which the titer is E0 according to the first aspect of the invention, and contacting the drug to be tested with the defective recombinant filovirus; and
(b) Detecting defective recombinant filovirus titer E1 after contact with the drug to be detected,
if E1 is more than or equal to 1E0, judging that the drug to be detected does not have the activity of resisting the filovirus or the activity of the drug to be detected is too weak to be used as the anti-filovirus drug.
If E1 is less than or equal to 0.8E0, judging that the drug to be detected has anti-filovirus activity.
In another preferred embodiment, if 0.2E0 < E1.ltoreq. 0.5E0, the drug to be tested is judged to have general anti-filovirus activity.
In another preferred example, if 0.1E0 is less than E1 and less than or equal to 0.2E0, the drug to be detected is judged to have stronger anti-filovirus activity.
In another preferred example, if E1 is less than or equal to 0.1E0, the drug to be tested is judged to have strong anti-filovirus activity.
In another preferred embodiment, the method may include comparing the detection values of a plurality of drugs to be tested to evaluate the effect of different drugs to be tested.
In a sixteenth aspect of the invention there is provided the use of a defective recombinant filovirus according to the first aspect of the invention for:
(a) Preparing filovirus particles in batches;
(b) Filovirus-host interaction studies; and/or
(c) And (5) establishing a transgenic complementary animal model.
In another preferred embodiment, the filovirus-host interaction study includes, but is not limited to: simulating the related immune response of host cells caused by filovirus infection, researching the avidity of the filovirus to target organs in vivo, and the like.
In another preferred embodiment, the studies applicable to the transgenic complementary animal model include, but are not limited to: study in vivo pathogenesis, test efficacy of anti-filovirus drugs or vaccines, and the like.
In a seventeenth aspect of the present invention there is provided a pharmaceutical composition according to the eighth aspect of the present invention or the use of a defective recombinant filovirus according to the first aspect of the present invention for the manufacture of a medicament for the prevention and/or treatment of a filovirus infection-associated disease.
In another preferred embodiment, the diseases associated with filovirus infection include, but are not limited to: nausea, vomiting, cramps, fever, dizziness, tiredness, weakness, sore throat, diarrhea, abdominal pain, chest pain, general rash, skin color changes, headache, general soreness, internal bleeding, external bleeding, blood clotting, hemorrhagic fever, liver failure, stroke, myocardial infarction, hypovolemic shock, multiple organ failure, death, and the like.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows rEBOVΔGP-Cre genomic cDNA vector and related eukaryotic expression plasmid.
Figure 2 shows the controlled expression of ebola virus GP protein.
FIG. 3 shows the life cycle of rEBOVΔGP-Cre recombinant viruses on complementing cells.
FIG. 4 shows the biological characteristics of rEBOVΔGP-Cre recombinant viruses.
FIG. 5 shows the use of rEBOVΔGP-Cre recombinant viruses in the detection of antiviral compound activity.
Detailed Description
The present inventors have made extensive and intensive studies and, as a result, have developed, for the first time, a defective recombinant filamentous virus in which the coding sequence of a key viral protein in the genome of the defective recombinant filamentous virus is replaced with the coding sequence of a recombinase. After the complementary cell is established through transduction of the regulatory expression vector, the regulatory expression vector in the complementary cell undergoes intramolecular rearrangement in the presence of the recombinase, thereby turning on the expression of the key viral protein on the vector. The vector or polynucleotide of the present invention is introduced into the complementary cell, and a defective recombinant filovirus having the same antigenic characteristics as the wild-type filovirus can be reconstituted.
The defective recombinant filovirus of the invention can not generate progeny infectious virus particles under conventional culture conditions and natural environment, and has no infectivity and pathogenicity, thus having better biological safety characteristics. Meanwhile, the recombinant filovirus of the invention can effectively proliferate in large quantity in complementary cells, and is easy to produce. The recombinant filovirus constructed by the method of the invention can completely simulate the related immune response of host cells caused by virus infection, and is a tool virus with extremely high practicability. The present invention has been completed on the basis of this finding.
Terminology
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein.
The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.
Ebola virus (EBOV)
Ebola virus genome (vRNA) is about 19kb in length, and encodes 7 viral proteins, nucleocapsid Protein (NP), VP30, VP35, VP40, glycoprotein (GP), VP24 and L proteins, respectively. Wherein the viral GP protein is a glycosylated protein. The virus particles are about 1 μm long, in the form of filaments, about 80 nm in diameter, branched, U-shaped, 6-shaped or annular, branched being more common. The envelope is inlaid with GP protein, which is a key virus structural protein responsible for virus recognition of host cells and virus particle invasion. GP is the only transmembrane protein of ebola virus responsible for receptor binding and membrane fusion and may also play an important role in the process of packaging mature virus out of cells.
Cre-lox recombination system
The Cre-lox recombination system is a site-specific gene recombination and rearrangement technique, and consists of a recombinase (Cre) and a DNA sequence specifically recognized by the recombinase, and lox sites. Wherein Cre recombinase is a site-specific recombinase capable of catalyzing site-specific recombination between two DNA recognition sites. Cre recombinase is derived from P1 phage and consists of 343 amino acids, and can specifically recognize Lox site. In addition to Cre, such recombinases are Flp (flip) and Dre (D6-specific recombinases).
The palindromic DNA site recognized by Cre recombinase, also called loxP (locus of X-over P1) site, is 34bp long and is characterized by the structure ATAACTTCGTATA-NNNTANNN-TATACGAAGTTAT (SEQ ID NO: 15). The 13 bases which are reversely complementary on both sides are the recognition sequence of Cre recombinase, and the 8 bases in the middle are the recombination occurrence positions, which also determine the direction of loxP. N represents a variable base, different base selections can form different Lox sites, in addition to wild-type loxP, loxN, lox2272, lox5171, etc., which are also recognized by Cre recombinase, but recombination can only occur between two Lox sites of identical sequence.
The result of recombination depends on the location and orientation of the loxP site. There are mainly several recombination modes: (1) Two loxP sites are positioned on the same DNA chain and have the same direction, and Cre recombinase knocks out sequences among loxP sites; (2) Two loxP sites are positioned on the same DNA chain and have opposite directions, and Cre recombinase induces sequence inversion between the loxP sites; (3) The two loxP sites are positioned on different DNA chains or chromosomes, and Cre recombinase induces the exchange or chromosomal translocation of the two DNA chains; (4) Four loxP sites are located on two DNA strands or chromosomes, respectively, and Cre recombinase induces sequence exchange between loxPs.
The Cre-lox system has the advantages of simple operation and high recombination rate, and is a powerful tool for in-vivo and in-vitro genetic operation. By using the Cre-lox system, a gene can be knocked out or expressed in a specific cell, tissue or whole organism, even at a specific time point, so that space-time specific operation on the specific gene can be realized.
Defective recombinant filoviruses of the invention
As used herein, the terms "defective virus", "defective recombinant virus", "defective filovirus", "defective recombinant filovirus", "recombinant virus" are used interchangeably and refer to a defective recombinant filovirus constructed according to the invention, in whose genome the coding sequence of a key viral protein is replaced by the coding sequence of a recombinase.
Typically, the genome structure of the defective recombinant filoviruses of the invention from 3 'to 5' is as shown in formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8 (I)
in the method, in the process of the invention,
each "-" is independently a bond or a nucleotide linking sequence;
z1 to Z8 are as described above; and at least one of Z3, Z4 and Z6 is a recombinant enzyme coding sequence.
It is understood that Z1 to Z8 are RNA sequences encoding the corresponding proteins.
A schematic representation of the genomic structure of a representative defective recombinant filovirus is shown in FIG. 3 as rEBOVΔGP-Cre vrNA.
The antigen characteristics of the defective recombinant virus are completely consistent with those of the wild type filovirus, but the defective recombinant virus does not express key viral proteins of replaced coding sequences in conventional culture conditions and natural environment, so that infectious progeny virus particles are not generated, and the biological safety is good.
Polynucleotides of the invention
As used herein, the terms "isolated polynucleotide", "polynucleotide sequence", and the like are used interchangeably and refer to a polynucleotide as described in the second aspect of the present invention, which may be DNA, RNA or cDNA.
Typically, the polynucleotide of the invention is selected from the group consisting of:
(a) A polynucleotide with a sequence shown as SEQ ID NO. 1;
(b) A polynucleotide having a nucleotide sequence having a homology of 95% (preferably 98%) or more with the sequence shown in SEQ ID NO. 1;
(c) A polynucleotide truncated at the 5 'and/or 3' end of the polynucleotide shown in SEQ ID NO. 1 or added with 1 to 99 (preferably 1 to 49, more preferably 1 to 9) nucleotides; and
(d) A polynucleotide complementary to the polynucleotide of any one of (a) - (c).
The sequence shown in SEQ ID NO. 1 is the full length of cDNA sequence of recombinant Ebola virus genome, and can be obtained by reverse transcription of the genome sequence of the recombinant Ebola virus.
A representative polynucleotide has a structure as shown in formula I:
Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8 (I)
in the method, in the process of the invention,
each "-" is independently a bond or a nucleotide linking sequence;
z1 to Z8 are as described above; and at least one of Z3, Z4 and Z6 is a recombinant enzyme coding sequence.
It is understood that Z1 to Z8 are DNA sequences encoding the corresponding proteins. Wherein Z1 is a sequence shown as SEQ ID NO. 2; z2 is a sequence shown as SEQ ID NO. 3; z3 is a sequence shown as SEQ ID NO. 4; z4 is a sequence shown as SEQ ID NO. 5; z5 is a sequence shown as SEQ ID NO. 6; z6 is a sequence shown as SEQ ID NO. 7; z7 is a sequence shown as SEQ ID NO. 8.
A schematic representation of a representative polynucleotide structure is shown in FIG. 1 in the rEBOV cDNA plasmid structure, with the addition of an HDV ribozyme sequence at the 3' end of the cDNA.
It will be appreciated that polynucleotides complementary to any of (a) to (c) also include polynucleotides which are reverse complementary to any of (a) to (c) and which pair U with a, when the polynucleotide is an RNA sequence, i.e. an RNA sequence transcribed from any of (a) to (c).
A schematic representation of a representative polynucleotide structure is shown in FIG. 3 as rEBOVΔGP-Cre vRNA.
The vector of the invention
As used herein, the term "vector" refers to a vector as described in the third aspect of the invention. It is to be understood that the carrier suitable for use in the present invention is not particularly limited.
A schematic representation of a representative vector structure is shown in FIG. 1 as ptBac6-rEBOV ΔGP-Cre, and the vector further comprises a human polymerase I promoter and a human polymerase I terminator.
Regulatory expression vectors
As used herein, the term "regulated expression vector" refers to a controlled expression vector that is regulated by expression of a recombinase, under appropriate conditions, to express a key viral protein, which corresponds to a defective recombinant filovirus in the following relationship:
If the coding sequence of the key viral protein (denoted by P1) in the genome of the defective recombinant virus is replaced by the coding sequence of the recombinase (denoted by P2), the corresponding regulatory expression vector is subjected to the expression regulation of P2, and when P2 exists, the expression of P1 is regulated.
Typically, the regulatory expression vector of the present invention has a structure as shown in formula II:
X1-X2-X3-X4-X5-X6-X7-X8 (II)
in the method, in the process of the invention,
each "-" is independently a bond or a nucleotide linking sequence;
x1 to X8 are as described above; and, the directions of X2 and X7 are opposite to induce the sequence between X2 and X7 to be rearranged in molecule, and the expression of the key viral protein on the vector is started (the representative process is shown in FIG. 2).
A schematic representation of a representative regulated expression vector structure is shown in the pDIO-GP plasmid of FIG. 2, which is regulated by the expression of recombinase Cre, and in the presence of this recombinase protein Cre, the GP protein is expressed in a controlled manner. Resistance genes, reporter genes, may also be included in the regulated expression vector to screen or characterize its expression in cells.
Complementary cells
As used herein, the term "complementing cell" refers to a eukaryotic cell into which the above-described regulatory expression vector is introduced, wherein "complementing" means that it complements the genome of the defective recombinant virus, and the meaning of "complementing" is as follows:
The defective recombinant viral genome does not express the key viral protein (denoted by P1) of the replaced coding sequence under conventional culture conditions and natural environment, but expresses the recombinase (denoted by P2) such as Cre protein; since the regulatory expression vector is introduced into the complementary cell, P1 can be expressed under control in the presence of P2.
Thus "complementary" is embodied in two ways: (1) The defective recombinant viral genome expresses P2, but P2 is not expressed in the complementing cells, and the complementing cells require expression of P2 to initiate expression of P1, the defective recombinant viral genome being "complementary" to the complementing cells in terms of expression of recombinase (P2); (2) The defective recombinant viral genome does not express P1, but when P2 derived from the defective recombinant viral genome is present in the complementing cell, the complementing cell is capable of expressing P1, and the defective recombinant viral genome is also "complementing" the complementing cell in terms of the expression of the key viral protein (P1).
When the defective recombinant virus grows and proliferates in the complementary cells, the viral protein (without P1) expressed by the defective recombinant virus genome will reconstruct with the key viral protein (P1) controlled to be expressed in the complementary cells, thereby forming the defective recombinant virus with self-limiting characteristic. The defective recombinant virus has viral proteins completely identical to the wild-type virus, but its genome does not express the key viral protein (P1) of the replaced coding sequence, and the introduction of P1 in the complete viral particles of the defective recombinant virus results from the reconstitution of P1 expressed by the complementing cells.
The expression vector may be regulated by introduction into the complementing cell by means of a liposome transfection reagent.
Representative eukaryotic cells into which the regulatory expression vector can be introduced are HEK293 cells, and representative complementary cells obtained by successfully introducing the regulatory expression vector and continuing to culture are HEK293-loxGP cells.
In a representative complementing cell HEK293-loxGP, since the complementing cell can continuously express the GP protein in the presence of the Cre protein, a defective recombinant virus capable of expressing the Cre protein but not the GP protein can effectively proliferate in the complementing cell and generate a large amount of progeny infectious virus particles through reconstitution with the GP protein, and on the basis, the defective recombinant virus can be stably passaged in the complementing cell system and prepared and purified in a large amount.
Compositions of the invention
As used herein, the term "composition" refers to a composition as described in the fifth aspect of the invention, comprising a vector as described in the third aspect of the invention and a regulatory expression vector, optionally comprising other viral protein expression vectors. The composition of the present invention can be used for preparing the defective recombinant filovirus according to the first aspect of the present invention, by introducing the regulatory expression vector into a eukaryotic cell, thereby culturing the cell to obtain the complementary cell, and introducing the vector according to the third aspect of the present invention (the other viral protein expression vector may also be introduced simultaneously) into the complementary cell, thereby culturing the cell to obtain the defective recombinant filovirus according to the first aspect of the present invention.
Host cells of the invention
It will be appreciated that the host cell of the invention contains a vector as described in the third aspect of the invention, or has incorporated into its genome a polynucleotide as described in the second aspect of the invention, but does not contain a regulatory expression vector.
The host cells of the invention do not express the critical viral protein (denoted by P1) of the replaced coding sequence, but instead express a recombinase (denoted by P2) such as the Cre protein, and thus do not produce infectious progeny viral particles. However, when a regulatory expression vector is introduced into the host cell of the present invention, the host cell can also be transformed into a complementing cell and P1 is expressed under control in the presence of P2, and upon reconstitution of P1 with other viral proteins infectious progeny viral particles can be formed.
Preparation method
The preparation of the defective recombinant filovirus according to the first aspect of the invention requires the introduction of mainly the following components:
(a) First component: a genome of a defective recombinant filovirus obtainable by introducing a vector according to the third aspect of the invention into a host cell, or by introducing a polynucleotide according to the second aspect of the invention into a host cell using other exogenous gene introduction methods;
(b) The second component: complementary cells, which can be constructed by introducing a regulatory expression vector into a suitable cell;
(c) Optionally a third component: other viral protein expression vectors.
It will be appreciated that the order and method of introducing the first component into the second component is not particularly limited, and the ultimate goal is to allow the genome of the defective recombinant filovirus to be present in the complementing cell, and thus allow the recombinant enzyme expressed by the genome of the defective recombinant filovirus to regulate the expression of the key viral protein in the complementing cell, thereby allowing the key viral protein to be reconstituted with other viral proteins within the complementing cell and assembled into defective recombinant filovirus particles having antigenic characteristics that are fully identical to those of the wild-type filovirus.
The method described in the sixth aspect of the present invention is an example of the production method of the present invention, and the production method of the present invention is not limited thereto, and any method is included in the production method of the present invention as long as the above-described first component can be introduced into the second component. For example, the above object can be achieved by introducing a regulatory expression vector into a host cell of the present invention (comprising a vector according to the third aspect of the present invention, or having incorporated into its genome a polynucleotide according to the second aspect of the present invention). There are no particular restrictions on the method and sequence of introduction of the regulatory expression vector, the vector according to the third aspect of the invention, the polynucleotide according to the second aspect of the invention, or other viral protein expression vector.
Standard reference substance
The defective recombinant filovirus of the invention can be used as a standard reference substance in detection methods or kits.
It will be appreciated that the detection method or kit suitable for using the defective recombinant filoviruses of the invention as a standard reference substance is not particularly limited and may include conventional techniques such as ELISA, PCR, etc.
Comparing the detection value of the sample to be detected with the corresponding detection value of the defective recombinant filovirus, the invention can qualitatively judge whether the sample to be detected contains the filovirus or quantitatively detect the content of the filovirus in the sample to be detected.
Pharmaceutical compositions or formulations and methods of administration
The invention provides a pharmaceutical composition or preparation for preventing and/or treating diseases related to filovirus infection.
The invention also provides a pharmaceutical composition or preparation for preventing and/or treating diseases related to filovirus infection. The pharmaceutical compositions or formulations of the invention comprise an inactivated or attenuated defective recombinant filovirus according to the first aspect of the invention, and may further comprise other drugs, together with their pharmaceutically acceptable carriers, diluents or excipients.
As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid or gel fillers that are suitable for use in humans or animals, and must be of sufficient purity and sufficiently low toxicity. "compatibility" means that the components of the pharmaceutical composition and the active ingredients of the drug and the combination thereof are blended with each other without significantly reducing the efficacy.
It will be appreciated that in the present invention, the pharmaceutically acceptable carrier is not particularly limited, and may be selected from materials commonly used in the art, or may be prepared by conventional methods, or may be commercially available. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., methylcellulose, ethylcellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifying agents (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), buffering agents, chelating agents, thickening agents, pH adjusting agents, transdermal enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostats, pyrogen-free water, etc.
In a preferred embodiment of the present invention, the pharmaceutical composition or formulation is in the form of a solid, liquid or semi-solid formulation.
In a preferred embodiment of the present invention, the pharmaceutical composition or the preparation is in the form of an oral preparation, an external preparation or an injection preparation.
Typically, the pharmaceutical composition or formulation is in the form of a tablet, injection, infusion, paste, gel, solution, microsphere or film.
The pharmaceutical composition or formulation should be compatible with the mode of administration. The pharmaceutical compositions or formulations of the present invention may also be used with (including before, during, or after) other co-therapeutic agents. When a pharmaceutical composition or formulation is used, a safe and effective amount of the drug is administered to a subject in need thereof (e.g., a human or non-human mammal), typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.
The main advantages of the invention include:
(1) The defective recombinant virus constructed by the method can not generate progeny infectious virus particles under conventional culture conditions and natural environment, has no infectivity and pathogenicity, and has better biological safety characteristics.
(2) The defective recombinant filovirus constructed by the invention can effectively proliferate and generate a large amount of progeny virus particles in a specific complementary cell line, so that the defective recombinant virus can be stably passaged in the specific complementary cell line and prepared and purified in a large amount, and has the characteristics of remarkable easiness in production and practicability.
(3) After the defective virus of the present invention infects host cells, biological processes such as virus invasion, genome release, virus mRNA transcription, virus protein expression, interaction with host factors, etc. can occur, and the defective virus is similar to wild type virus, so that the related immune response of the host cells caused after virus infection can be completely simulated.
(4) Because all viral proteins are expressed after the viral particles enter the step, the defective recombinant filovirus constructed by the invention can be widely applied to the research of virus-host interaction, has wide application range and can be used for a plurality of objects.
(5) When combined with reporter gene technology, the defective viruses and the complementary cells thereof of the invention have great application value in virology research, vaccine design and antiviral agent development, for example, the establishment of an anti-filovirus drug screening platform; infecting a common Cre gene activity reporter animal to study the in vivo nucleophilicity of the virus to the target organ; a transgenic complementary animal model is established for highly pathogenic viruses to study in vivo pathogenesis and test efficacy of antiviral drugs or vaccines, etc.
(6) The defective virus of the present invention has exactly the same components as the wild-type virus, in particular, the antigenic characteristics thereof are exactly identical to those of the wild-type ebola virus, and can be used for developing novel vaccines or as reference substances for clinical diagnostic tools and methods.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Sequence information
SEQ ID NO. 1 is the full length of the cDNA sequence of the recombinant Ebola virus genome;
SEQ ID NO. 2 and SEQ ID NO. 9 are respectively the DNA coding sequence and the amino acid sequence of the NP protein of the Ebola virus;
SEQ ID NO. 3 and SEQ ID NO. 10 are respectively the DNA coding sequence and the amino acid sequence of the Ebola virus VP35 protein;
SEQ ID NO. 4 and SEQ ID NO. 11 are respectively the DNA coding sequence and the amino acid sequence of the Ebola virus VP40 protein;
SEQ ID NO. 5 is the DNA coding sequence of recombinase Cre;
SEQ ID NO. 6 and SEQ ID NO. 12 are respectively the DNA coding sequence and the amino acid sequence of the Ebola virus VP30 protein;
SEQ ID NO. 7 and SEQ ID NO. 13 are respectively the DNA coding sequence and the amino acid sequence of the Ebola virus VP24 protein;
SEQ ID NO. 8 and SEQ ID NO. 14 are the DNA coding sequence and the amino acid sequence of the Ebola virus L protein, respectively;
SEQ ID NO. 15 is loxP site sequence recognized by Cre recombinase.
Example 1 preparation of defective recombinant Ebola Virus
(1) Defective recombinant Ebola virus genome cDNA vector with Cre gene and related expression vector And (3) counting:
the inventor uses Zaeel Ebola disease genome sequence as a template, replaces a GP protein coding sequence on a virus genome with a Cre protein coding sequence (Cre) to obtain a membrane protein defective recombinant Ebola virus genome (rEBOV delta GP-Cre) with Cre protein, utilizes a gene synthesis technology to synthesize a full-length genome cDNA fragment in a segmented mode, clones the full-length genome cDNA fragment onto a bacterial artificial chromosome vector (BAC vector), and introduces an HDV ribozyme sequence (HDV ribo.) at the 3' end of virus genome cDNA so as to promote the correct transcription of the end of virus genome cRNA.
As shown in FIG. 1, the addition of human polymerase I promoter (human poly-merase I promoter, hPrim.) and human polymerase I terminator (human polymerase I terminator, hPrim.) sequences upstream and downstream of the entire cDNA sequence allows the vector to be transcribed autonomously in human cells to produce defective viral genomic cRNA. Meanwhile, eukaryotic expression plasmids of virus structural proteins NP, VP35, L and VP30 are constructed and used for reconstructing recombinant virus particles.
(2) Establishment of complementary cells:
in order to establish a proliferative cell line complementary to the rEBOVΔGP-Cre virus genome, the inventors constructed a GP protein expression vector regulated by Cre protein expression. The regulatory expression vector has a GP protein expression sequence regulated by loxP site, and after being transduced into cells, the regulatory expression vector has no virus GP protein expression at first.
As shown in FIG. 2, once the functional Cre protein is present in the cell, the loxP site is recombined to initiate expression of the GP protein of the Ebola virus. HEK293-loxGP was established by transfecting HEK293 cells with this vector.
HEK293 cells were plated in 10cm dishes one day in advance, and 12. Mu.g of pDIO-GP plasmid was introduced into the plated HEK293 cells the next day using a liposome transfection reagent, and culture was continued for 48 hours to obtain HEK293-loxGP complementation cells.
(3) Preparation of GP-deficient recombinant ebola virus with Cre recombinase gene:
as shown in FIG. 3, the inventor introduces related recombinant Ebola virus vectors on HEK293-loxGP cells through transfection, and obtains the recombinant rEBOV delta GP-Cre recombinant Ebola virus on the cell line, thereby obtaining the defective recombinant Ebola virus with self-limiting characteristics, and the recombinant virus has good biological safety characteristics, no proliferation capability, no pathogenicity and no infectivity in common cultured cells or natural environment.
The rEBOV delta GP-Cre recombinant plasmid mixture (ptBac 6-rEBOV delta GP-Cre 5 mug, pNP 1 mug, pVP30 mug, pVP35 mug and pL 2 mug) is transduced into HEK293-loxGP complementary cells by using liposome reagent, and the cell culture supernatant is collected when the culture reaches 96 hours, and filtered by a 0.45 mu m filter, thus obtaining the rEBOV delta GP-Cre recombinant virus.
The rEBOV ΔGP-Cre recombinant virus obtained was diluted in a gradient (10 4 、10 5 、10 6 Three times of dilution), HEK293-loxGP cells are infected, the infection liquid is washed off after virus is added into the cells for infection for 2 hours, the fresh culture liquid is replaced for continuous culture of the cells, and cytopathic plaques caused by obvious virus proliferation can be seen as shown in FIG. 4A after 6 days; as shown in FIG. 4B, GFP protein expression in diseased cells was clearly observed using fluorescence microscopy; as shown in the electron microscope observation of FIG. 4C, rEBOV delta GP-Cre recombinant virus is similar to the reported wild type ebola virus in terms of virus particle size and morphology and can be used as a reference substanceThe kit is used for development and research work of ebola virus related detection reagents and detection methods.
EXAMPLE 2 evaluation of anti-Ebola Virus active Compounds
After the HEK293-loxGP cells are infected by rEBOVΔGP-Cre virus, the expression of intracellular viral proteins increases with time, indicating that the recombinant virus can proliferate and further infect in specific complementary cells, with complete viral life cycle.
HEK293-loxGP intracellular reporter gene signals have significant correlation with virus proliferation. By utilizing rEBOV delta GP-Cre virus and HEK293-loxGP cells which are complementary cells of the rEBOV delta GP-Cre virus, an in-vitro proliferation system of the Ebola virus can be established, and the antiviral activity of an active compound for inhibiting the proliferation of the Ebola virus can be detected on the system.
Using this method, the inventors detected the known anti-ebola virus compound remilast Wei Jituo remifene. As shown in the results of FIG. 5, the system can better reflect the in vitro antiviral inhibitory activity of the anti-Ebola virus compound by using the level of the viral genome in the culture supernatant or the level of the intracellular luciferase reporter gene as a detection index, and the activity detection result is equivalent to the reference value of the existing literature.
This result indicates that the rEBOV ΔGP-Cre recombinant virus is a suitable high throughput screening tool virus for anti-Ebola virus compounds.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Sequence listing
<110> Shanghai pharmaceutical institute of China academy of sciences
Wuhan Institute of Virology, Chinese Academy of Sciences
<120> preparation method and application of defective filovirus
<130> P2022-0076
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 18024
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> full-length cDNA sequence of recombinant Ebola Virus genome
<400> 1
cggacacaca aaaagaaaga agaattttta ggatcttttg tgtgcgaata actatgagga 60
agattaataa ttttcctctc attgaaattt atatcggaat ttaaattgaa attgttactg 120
taatcacacc tggtttgttt cagagccaca tcacaaagat agagaacaac ctaggtctcc 180
gaagggagca agggcatcag tgtgctcagt tgaaaatccc ttgtcaacac ctaggtctta 240
tcacatcaca agttccacct cagactctgc agggtgatcc aacaacctta atagaaacat 300
tattgttaaa ggacagcatt agttcacagt caaacaagca agattgagaa ttaaccttgg 360
ttttgaactt gaacacttag gggattgaag attcaacaac cctaaagctt ggggtaaaac 420
attggaaata gttaaaagac aaattgctcg gaatcacaaa attccgagta tggattctcg 480
tcctcagaaa atctggatgg cgccgagtct cactgaatct gacatggatt accacaagat 540
cttgacagca ggtctgtccg ttcaacaggg gattgttcgg caaagagtca tcccagtgta 600
tcaagtaaac aatcttgaag aaatttgcca acttatcata caggcctttg aagcaggtgt 660
tgattttcaa gagagtgcgg acagtttcct tctcatgctt tgtcttcatc atgcgtacca 720
gggagattac aaacttttct tggaaagtgg cgcagtcaag tatttggaag ggcacgggtt 780
ccgttttgaa gtcaagaagc gtgatggagt gaagcgcctt gaggaattgc tgccagcagt 840
atctagtgga aaaaacatta agagaacact tgctgccatg ccggaagagg agacaactga 900
agctaatgcc ggtcagtttc tctcctttgc aagtctattc cttccgaaat tggtagtagg 960
agaaaaggct tgccttgaga aggttcaaag gcaaattcaa gtacatgcag agcaaggact 1020
gatacaatat ccaacagctt ggcaatcagt aggacacatg atggtgattt tccgtttgat 1080
gcgaacaaat tttctgatca aatttctcct aatacaccaa gggatgcaca tggttgccgg 1140
gcatgatgcc aacgatgctg tgatttcaaa ttcagtggct caagctcgtt tttcaggctt 1200
attgattgtc aaaacagtac ttgatcatat cctacaaaag acagaacgag gagttcgtct 1260
ccatcctctt gcaaggaccg ccaaggtaaa aaatgaggtg aactccttta aggctgcact 1320
cagctccctg gccaagcatg gagagtatgc tcctttcgcc cgacttttga acctttctgg 1380
agtaaataat cttgagcatg gtcttttccc tcaactatcg gcaattgcac tcggagtcgc 1440
cacagcacac gggagtaccc tcgcaggagt aaatgttgga gaacagtatc aacaactcag 1500
agaggctgcc actgaggctg agaagcaact ccaacaatat gcagagtctc gcgaacttga 1560
ccatcttgga cttgatgatc aggaaaagaa aattcttatg aacttccatc agaaaaagaa 1620
cgaaatcagc ttccagcaaa caaacgctat ggtaactcta agaaaagagc gcctggccaa 1680
gctgacagaa gctatcactg ctgcgtcact gcccaaaaca agtggacatt acgatgatga 1740
tgacgacatt ccctttccag gacccatcaa tgatgacgac aatcctggcc atcaagatga 1800
tgatccgact gactcacagg atacgaccat tcccgatgtg gtggttgatc ccgatgatgg 1860
aagctacggc gaataccaga gttactcgga aaacggcatg aatgcaccag atgacttggt 1920
cctattcgat ctagacgagg acgacgagga cactaagcca gtgcctaata gatcgaccaa 1980
gggtggacaa cagaagaaca gtcaaaaggg ccagcatata gagggcagac agacacaatc 2040
caggccaatt caaaatgtcc caggccctca cagaacaatc caccacgcca gtgcgccact 2100
cacggacaat gacagaagaa atgaaccctc cggctcaacc agccctcgga tgctgacacc 2160
aattaacgaa gaggcagacc cactggacga tgccgacgac gagacgtcta gccttccgcc 2220
cttggagtca gatgatgaag agcaggacag ggacggaact tccaaccgca cacccactgt 2280
cgccccaccg gctcccgtat acagagatca ctctgaaaag aaagaactcc cgcaagacga 2340
gcaacaagat caggaccaca ctcaagaggc caggaaccag gacagtgaca acacccagtc 2400
agaacactct tttgaggaga tgtatcgcca cattctaaga tcacaggggc catttgatgc 2460
tgttttgtat tatcatatga tgaaggatga gcctgtagtt ttcagtacca gtgatggcaa 2520
agagtacacg tatccagact cccttgaaga ggaatatcca ccatggctca ctgaaaaaga 2580
ggctatgaat gaagagaata gatttgttac attggatggt caacaatttt attggccggt 2640
gatgaatcac aagaataaat tcatggcaat cctgcaacat catcagtgaa tgagcatgga 2700
acaatgggat gattcaaccg acaaatagct aacattaagt agtcaaggaa cgaaaacagg 2760
aagaattttt gatgtctaag gtgtgaatta ttatcacaat aaaagtgatt cttatttttg 2820
aatttaaagc tagcttatta ttactagccg tttttcaaag ttcaatttga gtcttaatgc 2880
aaataggcgt taagccacag ttatagccat aattgtaact caatattcta actagcgatt 2940
tatctaaatt aaattacatt atgcttttat aacttaccta ctagcctgcc caacatttac 3000
acgatcgttt tataattaag aaaaaactaa tgatgaagat taaaaccttc atcatcctta 3060
cgtcaattga attctctagc actcgaagct tattgtcttc aatgtaaaag aaaagctggt 3120
ctaacaagat gacaactaga acaaagggca ggggccatac tgcggccacg actcaaaacg 3180
acagaatgcc aggccctgag ctttcgggct ggatctctga gcagctaatg accggaagaa 3240
ttcctgtaag cgacatcttc tgtgatattg agaacaatcc aggattatgc tacgcatccc 3300
aaatgcaaca aacgaagcca aacccgaaga cgcgcaacag tcaaacccaa acggacccaa 3360
tttgcaatca tagttttgag gaggtagtac aaacattggc ttcattggct actgttgtgc 3420
aacaacaaac catcgcatca gaatcattag aacaacgcat tacgagtctt gagaatggtc 3480
taaagccagt ttatgatatg gcaaaaacaa tctcctcatt gaacagggtt tgtgctgaga 3540
tggttgcaaa atatgatctt ctggtgatga caaccggtcg ggcaacagca accgctgcgg 3600
caactgaggc ttattgggcc gaacatggtc aaccaccacc tggaccatca ctttatgaag 3660
aaagtgcgat tcggggtaag attgaatcta gagatgagac cgtccctcaa agtgttaggg 3720
aggcattcaa caatctaaac agtaccactt cactaactga ggaaaatttt gggaaacctg 3780
acatttcggc aaaggatttg agaaacatta tgtatgatca cttgcctggt tttggaactg 3840
ctttccacca attagtacaa gtgatttgta aattgggaaa agatagcaac tcattggaca 3900
tcattcatgc tgagttccag gccagcctgg ctgaaggaga ctctcctcaa tgtgccctaa 3960
ttcaaattac aaaaagagtt ccaatcttcc aagatgctgc tccacctgtc atccacatcc 4020
gctctcgcgg tgacattccc cgagcttgcc agaaaagctt gcgtccagtc ccaccatcgc 4080
ccaaaatcga tagaggttgg gtatgtgttt ttcagcttca agatggtaaa acacttggac 4140
tcaaaatttg agccaatctc ccttccctcc gaaagaggcg aataatagca gaggcttcaa 4200
ctgctgaact atagggtacg ttacattaat gatacacttg tgagtatcag ccctggataa 4260
tataagtcaa ttaaacgacc aagataaaat tgttcatatc tcgctagcag cttaaaatat 4320
aaatgtaata ggagctatat ctctgacagt attataatca attgttatta agtaacccaa 4380
accaaaagtg atgaagatta agaaaaacct acctcggctg agagagtgtt ttttcattaa 4440
ccttcatctt gtaaacgttg agcaaaattg ttaaaaatat gaggcgggtt atattgccta 4500
ctgctcctcc tgaatatatg gaggccatat accctgtcag gtcaaattca acaattgcta 4560
gaggtggcaa cagcaataca ggcttcctga caccggagtc agtcaatggg gacactccat 4620
cgaatccact caggccaatt gccgatgaca ccatcgacca tgccagccac acaccaggca 4680
gtgtgtcatc agcattcatc cttgaagcta tggtgaatgt catatcgggc cccaaagtgc 4740
taatgaagca aattccaatt tggcttcctc taggtgtcgc tgatcaaaag acctacagct 4800
ttgactcaac tacggccgcc atcatgcttg cttcatacac tatcacccat ttcggcaagg 4860
caaccaatcc acttgtcaga gtcaatcggc tgggtcctgg aatcccggat catcccctca 4920
ggctcctgcg aattggaaac caggctttcc tccaggagtt cgttcttccg ccagtccaac 4980
taccccagta tttcaccttt gatttgacag cactcaaact gatcacccaa ccactgcctg 5040
ctgcaacatg gaccgatgac actccaacag gatcaaatgg agcgttgcgt ccaggaattt 5100
catttcatcc aaaacttcgc cccattcttt tacccaacaa aagtgggaag aaggggaaca 5160
gtgccgatct aacatctccg gagaaaatcc aagcaataat gacttcactc caggacttta 5220
agatcgttcc aattgatcca accaaaaata tcatgggaat cgaagtgcca gaaactctgg 5280
tccacaagct gaccggtaag aaggtgactt ctaaaaatgg acaaccaatc atccctgttc 5340
ttttgccaaa gtacattggg ttggacccgg tggctccagg agacctcacc atggtaatca 5400
cacaggattg tgacacctgt cattctcctg caagtcttcc agctgtgatt gagaagtaat 5460
tgcaataatt gactcagatc cagttttata gaatcttctc agggatagtg ataacatcta 5520
tttagtaatc cgtccattag aggagacact tttaattgat caatatacta aaggtgcttt 5580
acaccattgt cttttttctc tcctaaatgt agaacttaac aaaagactca taatatactt 5640
gtttttaaag gattgattga tgaaagatca taactaataa cattacaaat aatcctacta 5700
taatcaatac ggtgattcaa atgttaatct ttctcattgc acatactttt tgcccttatc 5760
ctcaaattgc ctgcatgctt acatctgagg atagccagtg tgacttggat tggaaatgtg 5820
gagaaaaaat cgggacccat ttctaggttg ttcacaatcc aagtacagac attgcccttc 5880
taattaagaa aaaatcggcg atgaagatta agccgacagt gagcgtaatc ttcatctctc 5940
ttagattatt tgttttccag agtaggggtc gtcaggtcct tttcaatcgt gtaaccaaaa 6000
taaactccac tagaaggata ttgtggggca acaacacaat gagcggccct cccaagaaga 6060
agcggaaggt agaagacccg ggaggtggga gcggcagcaa cctgctgaca gtgcaccaga 6120
acctgcccgc cctgcctgtg gatgccacca gcgatgaggt gagaaagaat ctgatggaca 6180
tgtttaggga tagacaggct tttagcgagc acacctggaa gatgctgctg agcgtgtgta 6240
ggtcctgggc cgcctggtgt aagctgaata atagaaagtg gttccctgcc gagcccgagg 6300
acgtgagaga ctacctgctg tacctccagg cccggggcct ggccgtgaag accatccagc 6360
agcacctggg ccagctgaat atgctgcaca ggaggagcgg cctgcccagg ccatccgaca 6420
gcaacgccgt gtccctggtc atgagaagaa tcagaaagga gaatgtggat gccggcgaga 6480
gagccaagca ggccctggct tttgagagaa ccgatttcga tcaggtgagg tccctgatgg 6540
agaatagcga cagatgccag gacatcagga atctggcctt tctgggcatc gcctacaata 6600
ccctgctcag gatcgccgag atcgccagaa tcagagtgaa ggacatcagc agaaccgacg 6660
gcggcaggat gctgatccac atcggcagaa ccaagaccct ggtgagcaca gccggcgtgg 6720
agaaggccct gagcctggga gtgaccaagc tggtggagag gtggatctcc gtgagcggcg 6780
tggccgacga ccccaataac tacctgtttt gtagggtgag gaagaacggc gtggccgccc 6840
ccagcgctac aagccaactg agcaccagag ccctggaggg catctttgag gccacacaca 6900
ggctgatcta cggggccaag gacgacagcg gccagagata cctggcctgg agcggccact 6960
ccgccagagt gggagccgct agagatatgg ccagggccgg cgtgagcatc cccgagatca 7020
tgcaggccgg cggctggacc aatgtgaata tcgtgatgaa ctacatcaga aacctggatt 7080
ccgagacagg cgctatggtt agactgctgg aggatggcta gacgcgtttt ttcttcagat 7140
tgcttcatgg aaaagctcag cctcaaatca atgaaaccag gatttaatta tatggattac 7200
ttgaatctaa gattacttga caaatgataa tataatacac tggagcttta aacatagcca 7260
atgtgattct aactccttta aactcacagt taatcataaa caaggtttga catcaatcta 7320
gttatctctt tgagaatgat aaacttgatg aagattaaga aaaaggtaat ctttcgatta 7380
tctttaatct tcatccttga ttctacaatc atgacagttg tctttagtga caagggaaag 7440
aagccttttt attaagttgt aataatcaga tctgcgaacc ggtagagttt agttgcaacc 7500
taacacacat aaagcattgg tcaaaaagtc aatagaaatt taaacagtga gtggagacaa 7560
cttttaaatg gaagcttcat atgagagagg acgcccacga gctgccagac agcattcaag 7620
ggatggacac gaccaccatg ttcgagcacg atcatcatcc agagagaatt atcgaggtga 7680
gtaccgtcaa tcaaggagcg cctcacaagt gcgcgttcct actgtatttc ataagaagag 7740
agttgaacca ttaacagttc ctccagcacc taaagacata tgtccgacct tgaaaaaagg 7800
atttttgtgt gacagtagtt tttgcaaaaa agatcaccag ttggagagtt taactgatag 7860
ggaattactc ctactaatcg cccgtaagac ttgtggatca gtagaacaac aattaaatat 7920
aactgcaccc aaggactcgc gcttagcaaa tccaacggct gatgatttcc agcaagagga 7980
aggtccaaaa attaccttgt tgacactgat caagacggca gaacactggg cgagacaaga 8040
catcagaacc atagaggatt caaaattaag agcattgttg actctatgtg ctgtgatgac 8100
gaggaaattc tcaaaatccc agctgagtct tttatgtgag acacacctaa ggcgcgaggg 8160
gcttgggcaa gatcaggcag aacccgttct cgaagtatat caacgattac acagtgataa 8220
aggaggcagt tttgaagctg cactatggca acaatgggac cgacaatccc taattatgtt 8280
tatcactgca ttcttgaata ttgctctcca gttaccgtgt gaaagttctg ctgtcgttgt 8340
ttcagggtta agaacattgg ttcctcaatc agataatgag gaagcttcaa ccaaccccgg 8400
cacatgctca tggtctgatg agggtacccc ttaataaggc tgactaaaac actatataac 8460
cttctacttg atcacaatac tccgtatacc tatcatcata tatttaatca agacgatatc 8520
ctttaaaact tattcagtac tataatcact ctcgtttcaa attaataaga tgtgcatgat 8580
tgccctaata tatgaagagg tatgatacaa ccctaacagt gatcaaagaa aatcataatc 8640
tcgtatcgct cgtaatataa cctgccaagc atacctcttg cacaaagtga ttcttgtaca 8700
caaataatgt tttactctac aggaggtagc aacgatccat cccatcaaaa aataagtatt 8760
tcatgactta ctaatgatct cttaaaatat taagaaaaac tgacggaaca taaattcttt 8820
atgcttcaag ctgtggagga ggtgtttggt attggctatt gttatattac aatcaataac 8880
aagcttgtaa aaatattgtt cttgtttcaa gaggtagatt gtgaccggaa atgctaaact 8940
aatgatgaag attaatgcgg aggtctgata agaataaacc ttattattca gattaggccc 9000
caagaggcat tcttcatctc cttttagcaa agtactattt cagggtagtc caattagtgg 9060
cacgtctttt agctgtatat cagtcgcccc tgagatacgc cacaaaagtg tctctaagct 9120
aaattggtct gtacacatcc catacattgt attaggggca ataatatcta attgaactta 9180
gccgtttaaa atttagtgca taaatctggg ctaacaccac caggtcaact ccattggctg 9240
aaaagaagct tacctacaac gaacatcact ttgagcgccc tcacaattaa aaaataggaa 9300
cgtcgttcca acaatcgagc gcaaggtttc aaggttgaac tgagagtgtc tagacaacaa 9360
aatattgata ctccagacac caagcaagac ctgagaaaaa accatggcta aagctacggg 9420
acgatacaat ctaatatcgc ccaaaaagga cctggagaaa ggggttgtct taagcgacct 9480
ctgtaacttc ttagttagcc aaactattca ggggtggaag gtttattggg ctggtattga 9540
gtttgatgtg actcacaaag gaatggccct attgcataga ctgaaaacta atgactttgc 9600
ccctgcatgg tcaatgacaa ggaatctctt tcctcattta tttcaaaatc cgaattccac 9660
aattgaatca ccgctgtggg cattgagagt catccttgca gcagggatac aggaccagct 9720
gattgaccag tctttgattg aacccttagc aggagccctt ggtctgatct ctgattggct 9780
gctaacaacc aacactaacc atttcaacat gcgaacacaa cgtgtcaagg aacaattgag 9840
cctaaaaatg ctgtcgttga ttcgatccaa tattctcaag tttattaaca aattggatgc 9900
tctacatgtc gtgaactaca acggattgtt gagcagtatt gaaattggaa ctcaaaatca 9960
tacaatcatc ataactcgaa ctaacatggg ttttctggtg gagctccaag aacccgacaa 10020
atcggcaatg aaccgcatga agcctgggcc ggcgaaattt tccctccttc atgagtccac 10080
actgaaagca tttacacaag ggagctcgac acgaatgcaa agtttgattc ttgaatttaa 10140
tagctctctt gctatctaac taaggtagaa tacttcatat tgagctaact catatatgct 10200
gactcaatag ttatcttgac atctctgctt tcataatcag atatataagc ataataaata 10260
aatactcata tttcttgata atttgtttaa ccacagataa atcctcactg taagccagct 10320
tccaagttga cacccttaca aaaaccagga ctcagaatcc ctcaaacaag agattccaag 10380
acaacatcat agaattgctt tattatatga ataagcattt tatcaccaga aatcctatat 10440
actaaatggt taattgtaac tgaacccgca ggtcacatgt gttaggtttc acagattcta 10500
tatattacta actctatact cgtaattaac attagataag tagattaaga aaaaagcctg 10560
aggaagatta agaaaaactg cttattgggt ctttccgtgt tttagatgaa gcagttgaaa 10620
ttcttcctct tgatattaaa tggctacaca acatacccaa tacccagacg ctaggttatc 10680
atcaccaatt gtattggacc aatgtgacct agtcactcga gcttgcgggt tatattcatc 10740
atactccctt aatccgcaac tacgcaactg taaactcccg aaacatatct accgtttgaa 10800
atacgatgta actgttacca agttcttgag tgatgtacca gtggcgacat tgcccataga 10860
tttcatagtc ccagttcttc tcaaggcact gtcaggcaat ggattctgtc ctgttgagcc 10920
gcggtgccaa cagttcttag atgaaatcat taagtacaca atgcaagatg ctctcttctt 10980
gaaatattat ctcaaaaatg tgggtgctca agaagactgt gttgatgaac actttcaaga 11040
gaaaatctta tcttcaattc agggcaatga atttttacat caaatgtttt tctggtatga 11100
tctggctatt ttaactcgaa ggggtagatt aaatcgagga aactctagat caacatggtt 11160
tgttcatgat gatttaatag acatcttagg ctatggggac tatgtttttt ggaagatccc 11220
aatttcaatg ttaccactga acacacaagg aatcccccat gctgctatgg actggtatca 11280
ggcatcagta ttcaaagaag cggttcaagg gcatacacac attgtttctg tttctactgc 11340
cgacgtgttg ataatgtgca aagatttaat tacatgtcga ttcaacacaa ctctaatctc 11400
aaaaatagca gagattgagg acccagtttg ttctgattat cccaatttta agattgtgtc 11460
tatgctttac cagagcggag attacttact ctccatatta gggtctgatg ggtataaaat 11520
tattaagttc ctcgaaccat tgtgcttggc caaaattcaa ttatgctcaa agtacactga 11580
gaggaagggc cgattcttaa cacaaatgca tttagctgta aatcacaccc tagaagaaat 11640
tacagaaatg cgtgcactaa agccttcaca ggctcaaaag atccgtgagt tccatagaac 11700
attgataagg ctggagatga cgccacaaca actttgtgag ctattttcca ttcaaaaaca 11760
ctgggggcat cctgtgctac atagtgaaac agcaatccaa aaagttaaaa aacatgctac 11820
ggtgctaaaa gcattacgcc ctatagtgat tttcgagaca tactgtgttt ttaaatatag 11880
tattgccaaa cattattttg atagtcaagg atcttggtac agtgttactt cagataggaa 11940
tctaacaccg ggtcttaatt cttatatcaa aagaaatcaa ttccctccgt tgccaatgat 12000
taaagaacta ctatgggaat tttaccacct tgaccaccct ccacttttct caaccaaaat 12060
tattagtgac ttaagtattt ttataaaaga cagagctacc gcagtagaaa ggacatgctg 12120
ggatgcagta ttcgagccta atgttctagg atataatcca cctcacaaat ttagtactaa 12180
acgtgtaccg gaacaatttt tagagcaaga aaacttttct attgagaatg ttctttccta 12240
cgcacaaaaa ctggagtatc tactaccaca atatcggaac ttttctttct cattgaaaga 12300
gaaagagttg aatgtaggta gaaccttcgg aaaattgcct tatccgactc gcaatgttca 12360
aacactttgt gaagctctgt tagctgatgg tcttgctaaa gcatttccta gcaatatgat 12420
ggtagttacg gaacgtgagc aaaaagaaag cttattgcat caagcatcat ggcaccacac 12480
aagtgatgat tttggtgaac atgccacagt tagagggagt agctttgtaa ctgatttaga 12540
gaaatacaat cttgcattta gatatgagtt tacagcacct tttatagaat attgcaaccg 12600
ttgctatggt gttaagaatg tttttaattg gatgcattat acaatcccac agtgttatat 12660
gcatgtcagt gattattata atccaccaca taacctcaca ctggagaatc gagacaaccc 12720
ccccgaaggg cctagttcat acaggggtca tatgggaggg attgaaggac tgcaacaaaa 12780
actctggaca agtatttcat gtgctcaaat ttctttagtt gaaattaaga ctggttttaa 12840
gttacgctca gctgtgatgg gtgacaatca gtgcattact gttttatcag tcttcccctt 12900
agagactgac gcagacgagc aggaacagag cgccgaagac aatgcagcga gggtggccgc 12960
cagcctagca aaagttacaa gtgcctgtgg aatcttttta aaacctgatg aaacatttgt 13020
acattcaggt tttatctatt ttggaaaaaa acaatatttg aatggggtcc aattgcctca 13080
gtcccttaaa acggctacaa gaatggcacc attgtctgat gcaatttttg atgatcttca 13140
agggaccctg gctagtatag gcactgcttt tgagcgatcc atctctgaga cacgacatat 13200
ctttccttgc aggataaccg cagctttcca tacgtttttt tcggtgagaa tcttgcaata 13260
tcatcatctc gggttcaata aaggttttga ccttggacag ttgacactcg gcaaacctct 13320
ggatttcgga acaatatcat tggcactagc ggtcccgcag gtgcttggag ggttatcctt 13380
cttgaatcct gagaaatgtt tctaccggaa tctaggagat ccagttacct caggcttatt 13440
ccagttaaaa acttatctcc gaatgattga gatggatgat ttattcttac ctttaattgc 13500
gaagaaccct gggaactgca ctgccattga ctttgtgcta aatcctagcg gattaaatgt 13560
ccctgggtcg caagacttaa cttcatttct gcgccagatt gtacgcagga ccatcaccct 13620
aagtgcgaaa aacaaactta ttaatacctt atttcatgcg tcagctgact tcgaagacga 13680
aatggtttgt aaatggctat tatcatcaac tcctgttatg agtcgttttg cggccgatat 13740
cttttcacgc acgccgagcg ggaagcgatt gcaaattcta ggatacctgg aaggaacacg 13800
cacattatta gcctctaaga tcatcaacaa taatacagag acaccggttt tggacagact 13860
gaggaaaata acattgcaaa ggtggagcct atggtttagt tatcttgatc attgtgataa 13920
tatcctggcg gaggctttaa cccaaataac ttgcacagtt gatttagcac agattctgag 13980
ggaatattca tgggctcata ttttagaggg aagacctctt attggagcca cactcccatg 14040
tatgattgag caattcaaag tgttttggct gaaaccctac gaacaatgtc cgcagtgttc 14100
aaatgcaaag caaccaggtg ggaaaccatt cgtgtcagtg gcagtcaaga aacatattgt 14160
tagtgcatgg ccgaacgcat cccgaataag ctggactatc ggggatggaa tcccatacat 14220
tggatcaagg acagaagata agataggaca acctgctatt aaaccaaaat gtccctcagc 14280
agccttaaga gaggccattg aattggcgtc ccgtttaaca tgggtaactc aaggcagttc 14340
gaacagtgac ttgctaataa aaccattttt ggaagcacga gtaaatttaa gtgttcaaga 14400
aatacttcaa atgacccctt cacattactc aggaaatatt gttcacaggt acaacgatca 14460
atacagtcct cattctttca tggccaatcg tatgagtaat tcagcaacgc gattgattgt 14520
ttctacaaac actttaggtg agttttcagg aggtggccag tctgcacgcg acagcaatat 14580
tattttccag aatgttataa attatgcagt tgcactgttc gatattaaat ttagaaacac 14640
tgaggctaca gatatccaat ataatcgtgc tcaccttcat ctaactaagt gttgcacccg 14700
cgaagtacca gctcagtatt taacatacac atctacattg gatttagatt taacaagata 14760
ccgagaaaac gaattgattt atgacagtaa tcctctaaaa ggaggactca attgcaatat 14820
ctcattcgat aatccatttt tccaaggtaa acggctgaac attatagaag atgatcttat 14880
tcgactgcct cacttatctg gatgggagct agccaagacc atcatgcaat caattatttc 14940
agatagcaac aattcatcta cagacccaat tagcagtgga gaaacaagat cattcactac 15000
ccatttctta acttatccca agataggact tctgtacagt tttggggcct ttgtaagtta 15060
ttatcttggc aatacaattc ttcggactaa gaaattaaca cttgacaatt ttttatatta 15120
cttaactact caaattcata atctaccaca tcgctcattg cgaatactta agccaacatt 15180
caaacatgca agcgttatgt cacggttaat gagtattgat cctcattttt ctatttacat 15240
aggcggtgct gcaggtgaca gaggactctc agatgcggcc aggttatttt tgagaacgtc 15300
catttcatct tttcttacat ttgtaaaaga atggataatt aatcgcggaa caattgtccc 15360
tttatggata gtatatccgc tagagggtca aaacccaaca cctgtgaata attttctcta 15420
tcagatcgta gaactgctgg tgcatgattc atcaagacaa caggctttta aaactaccat 15480
aagtgatcat gtacatcctc acgacaatct tgtttacaca tgtaagagta cagccagcaa 15540
tttcttccat gcatcattgg cgtactggag gagcagacac agaaacagca accgaaaata 15600
cttggcaaga gactcttcaa ctggatcaag cacaaacaac agtgatggtc atattgagag 15660
aagtcaagaa caaaccacca gagatccaca tgatggcact gaacggaatc tagtcctaca 15720
aatgagccat gaaataaaaa gaacgacaat tccacaagaa aacacgcacc agggtccgtc 15780
gttccagtcc tttctaagtg actctgcttg tggtacagca aatccaaaac taaatttcga 15840
taggtcgaga cacaatgtga aatttcagga tcataactcg gcatccaaga gggaaggtca 15900
tcaaataatc tcacaccgtc tagtcctacc tttctttaca ttatctcaag ggacacgcca 15960
attaacgtca tccaatgagt cacaaaccca agacgagata tcaaagtact tacggcaatt 16020
gagatccgtc attgatacca cagtttattg tagatttacc ggtatagtct cgtccatgca 16080
ttacaaactt gatgaggtcc tttgggaaat agagagtttc aagtcggctg tgacgctagc 16140
agagggagaa ggtgctggtg ccttactatt gattcagaaa taccaagtta agaccttatt 16200
tttcaacacg ctagctactg agtccagtat agagtcagaa atagtatcag gaatgactac 16260
tcctaggatg cttctacctg ttatgtcaaa attccataat gaccaaattg agattattct 16320
taacaactca gcaagccaaa taacagacat aacaaatcct acttggttta aagaccaaag 16380
agcaaggcta cctaagcaag tcgaggttat aaccatggat gcagagacaa cagagaatat 16440
aaacagatcg aaattgtacg aagctgtata taaattgatc ttacaccata ttgatcctag 16500
cgtattgaaa gcagtggtcc ttaaagtctt tctaagtgat actgagggta tgttatggct 16560
aaatgataat ttagccccgt tttttgccac tggttattta attaagccaa taacgtcaag 16620
tgctagatct agtgagtggt atctttgtct gacgaacttc ttatcaacta cacgtaagat 16680
gccacaccaa aaccatctca gttgtaaaca ggtaatactt acggcattgc aactgcaaat 16740
tcaacgaagc ccatactggc taagtcattt aactcagtat gctgactgtg agttacattt 16800
aagttatatc cgccttggtt ttccatcatt agagaaagta ctataccaca ggtataacct 16860
cgtcgattca aaaagaggtc cactagtctc tatcactcag cacttagcac atcttagagc 16920
agagattcga gaattaacta atgattataa tcaacagcga caaagtcgga ctcaaacata 16980
tcactttatt cgtactgcaa aaggacgaat cacaaaacta gtcaatgatt atttaaaatt 17040
ctttcttatt gtgcaagcat taaaacataa tgggacatgg caagctgagt ttaagaaatt 17100
accagagttg attagtgtgt gcaataggtt ctaccatatt agagattgca attgtgaaga 17160
acgtttctta gttcaaacct tatatttaca tagaatgcag gattctgaag ttaagcttat 17220
cgaaaggctg acagggcttc tgagtttatt tccggatggt ctctacaggt ttgatggatc 17280
ctgaattacc gtgcatagta tcctgatact tgcaaaggtt ggttattaac atacagatta 17340
taaaaaactc ataaattgct ctcatacatc atattgatct aatctcaata aacaactatt 17400
taaataacga aaggagtccc tatattatat actatattta gcctctctcc ctgcgtgata 17460
atcaaaaaat tcacaatgca gcatgtgtga catattactg ccgcaatgaa tttaacgcaa 17520
cataataaac tctgcactct ttataattaa gctttaacga aaggtctggg ctcatattgt 17580
tattgatata ataatgttgt atcaatatcc tgtcagatgg aatagtgttt tggttgataa 17640
cacaacttct taaaacaaaa ttgatcttta agattaagtt ttttataatt atcattactt 17700
taatttgtcg ttttaaaaac ggtgatagcc ttaatctttg tgtaaaataa gagattaggt 17760
gtaataacct taacattttt gtctagtaag ctactatttc atacagaatg ataaaattaa 17820
aagaaaaggc aggactgtaa aatcagaaat accttcttta caatatagca gactagataa 17880
taatcttcgt gttaatgata attaagacat tgaccacgct catcagaagg ctcgccagaa 17940
taaacgttgc aaaaaggatt cctggaaaaa tggtcgcaca caaaaattta aaaataaatc 18000
tatttcttct tttttgtgtg tcca 18024
<210> 2
<211> 2220
<212> DNA
<213> Ebola virus (Ebola virus)
<400> 2
atggattctc gtcctcagaa aatctggatg gcgccgagtc tcactgaatc tgacatggat 60
taccacaaga tcttgacagc aggtctgtcc gttcaacagg ggattgttcg gcaaagagtc 120
atcccagtgt atcaagtaaa caatcttgaa gaaatttgcc aacttatcat acaggccttt 180
gaagcaggtg ttgattttca agagagtgcg gacagtttcc ttctcatgct ttgtcttcat 240
catgcgtacc agggagatta caaacttttc ttggaaagtg gcgcagtcaa gtatttggaa 300
gggcacgggt tccgttttga agtcaagaag cgtgatggag tgaagcgcct tgaggaattg 360
ctgccagcag tatctagtgg aaaaaacatt aagagaacac ttgctgccat gccggaagag 420
gagacaactg aagctaatgc cggtcagttt ctctcctttg caagtctatt ccttccgaaa 480
ttggtagtag gagaaaaggc ttgccttgag aaggttcaaa ggcaaattca agtacatgca 540
gagcaaggac tgatacaata tccaacagct tggcaatcag taggacacat gatggtgatt 600
ttccgtttga tgcgaacaaa ttttctgatc aaatttctcc taatacacca agggatgcac 660
atggttgccg ggcatgatgc caacgatgct gtgatttcaa attcagtggc tcaagctcgt 720
ttttcaggct tattgattgt caaaacagta cttgatcata tcctacaaaa gacagaacga 780
ggagttcgtc tccatcctct tgcaaggacc gccaaggtaa aaaatgaggt gaactccttt 840
aaggctgcac tcagctccct ggccaagcat ggagagtatg ctcctttcgc ccgacttttg 900
aacctttctg gagtaaataa tcttgagcat ggtcttttcc ctcaactatc ggcaattgca 960
ctcggagtcg ccacagcaca cgggagtacc ctcgcaggag taaatgttgg agaacagtat 1020
caacaactca gagaggctgc cactgaggct gagaagcaac tccaacaata tgcagagtct 1080
cgcgaacttg accatcttgg acttgatgat caggaaaaga aaattcttat gaacttccat 1140
cagaaaaaga acgaaatcag cttccagcaa acaaacgcta tggtaactct aagaaaagag 1200
cgcctggcca agctgacaga agctatcact gctgcgtcac tgcccaaaac aagtggacat 1260
tacgatgatg atgacgacat tccctttcca ggacccatca atgatgacga caatcctggc 1320
catcaagatg atgatccgac tgactcacag gatacgacca ttcccgatgt ggtggttgat 1380
cccgatgatg gaagctacgg cgaataccag agttactcgg aaaacggcat gaatgcacca 1440
gatgacttgg tcctattcga tctagacgag gacgacgagg acactaagcc agtgcctaat 1500
agatcgacca agggtggaca acagaagaac agtcaaaagg gccagcatat agagggcaga 1560
cagacacaat ccaggccaat tcaaaatgtc ccaggccctc acagaacaat ccaccacgcc 1620
agtgcgccac tcacggacaa tgacagaaga aatgaaccct ccggctcaac cagccctcgg 1680
atgctgacac caattaacga agaggcagac ccactggacg atgccgacga cgagacgtct 1740
agccttccgc ccttggagtc agatgatgaa gagcaggaca gggacggaac ttccaaccgc 1800
acacccactg tcgccccacc ggctcccgta tacagagatc actctgaaaa gaaagaactc 1860
ccgcaagacg agcaacaaga tcaggaccac actcaagagg ccaggaacca ggacagtgac 1920
aacacccagt cagaacactc ttttgaggag atgtatcgcc acattctaag atcacagggg 1980
ccatttgatg ctgttttgta ttatcatatg atgaaggatg agcctgtagt tttcagtacc 2040
agtgatggca aagagtacac gtatccagac tcccttgaag aggaatatcc accatggctc 2100
actgaaaaag aggctatgaa tgaagagaat agatttgtta cattggatgg tcaacaattt 2160
tattggccgg tgatgaatca caagaataaa ttcatggcaa tcctgcaaca tcatcagtga 2220
<210> 3
<211> 1023
<212> DNA
<213> Ebola virus (Ebola virus)
<400> 3
atgacaacta gaacaaaggg caggggccat actgcggcca cgactcaaaa cgacagaatg 60
ccaggccctg agctttcggg ctggatctct gagcagctaa tgaccggaag aattcctgta 120
agcgacatct tctgtgatat tgagaacaat ccaggattat gctacgcatc ccaaatgcaa 180
caaacgaagc caaacccgaa gacgcgcaac agtcaaaccc aaacggaccc aatttgcaat 240
catagttttg aggaggtagt acaaacattg gcttcattgg ctactgttgt gcaacaacaa 300
accatcgcat cagaatcatt agaacaacgc attacgagtc ttgagaatgg tctaaagcca 360
gtttatgata tggcaaaaac aatctcctca ttgaacaggg tttgtgctga gatggttgca 420
aaatatgatc ttctggtgat gacaaccggt cgggcaacag caaccgctgc ggcaactgag 480
gcttattggg ccgaacatgg tcaaccacca cctggaccat cactttatga agaaagtgcg 540
attcggggta agattgaatc tagagatgag accgtccctc aaagtgttag ggaggcattc 600
aacaatctaa acagtaccac ttcactaact gaggaaaatt ttgggaaacc tgacatttcg 660
gcaaaggatt tgagaaacat tatgtatgat cacttgcctg gttttggaac tgctttccac 720
caattagtac aagtgatttg taaattggga aaagatagca actcattgga catcattcat 780
gctgagttcc aggccagcct ggctgaagga gactctcctc aatgtgccct aattcaaatt 840
acaaaaagag ttccaatctt ccaagatgct gctccacctg tcatccacat ccgctctcgc 900
ggtgacattc cccgagcttg ccagaaaagc ttgcgtccag tcccaccatc gcccaaaatc 960
gatagaggtt gggtatgtgt ttttcagctt caagatggta aaacacttgg actcaaaatt 1020
tga 1023
<210> 4
<211> 981
<212> DNA
<213> Ebola virus (Ebola virus)
<400> 4
atgaggcggg ttatattgcc tactgctcct cctgaatata tggaggccat ataccctgtc 60
aggtcaaatt caacaattgc tagaggtggc aacagcaata caggcttcct gacaccggag 120
tcagtcaatg gggacactcc atcgaatcca ctcaggccaa ttgccgatga caccatcgac 180
catgccagcc acacaccagg cagtgtgtca tcagcattca tccttgaagc tatggtgaat 240
gtcatatcgg gccccaaagt gctaatgaag caaattccaa tttggcttcc tctaggtgtc 300
gctgatcaaa agacctacag ctttgactca actacggccg ccatcatgct tgcttcatac 360
actatcaccc atttcggcaa ggcaaccaat ccacttgtca gagtcaatcg gctgggtcct 420
ggaatcccgg atcatcccct caggctcctg cgaattggaa accaggcttt cctccaggag 480
ttcgttcttc cgccagtcca actaccccag tatttcacct ttgatttgac agcactcaaa 540
ctgatcaccc aaccactgcc tgctgcaaca tggaccgatg acactccaac aggatcaaat 600
ggagcgttgc gtccaggaat ttcatttcat ccaaaacttc gccccattct tttacccaac 660
aaaagtggga agaaggggaa cagtgccgat ctaacatctc cggagaaaat ccaagcaata 720
atgacttcac tccaggactt taagatcgtt ccaattgatc caaccaaaaa tatcatggga 780
atcgaagtgc cagaaactct ggtccacaag ctgaccggta agaaggtgac ttctaaaaat 840
ggacaaccaa tcatccctgt tcttttgcca aagtacattg ggttggaccc ggtggctcca 900
ggagacctca ccatggtaat cacacaggat tgtgacacct gtcattctcc tgcaagtctt 960
ccagctgtga ttgagaagta a 981
<210> 5
<211> 1023
<212> DNA
<213> P1 phage (Bacteriophage P1)
<400> 5
agcaacctgc tgacagtgca ccagaacctg cccgccctgc ctgtggatgc caccagcgat 60
gaggtgagaa agaatctgat ggacatgttt agggatagac aggcttttag cgagcacacc 120
tggaagatgc tgctgagcgt gtgtaggtcc tgggccgcct ggtgtaagct gaataataga 180
aagtggttcc ctgccgagcc cgaggacgtg agagactacc tgctgtacct ccaggcccgg 240
ggcctggccg tgaagaccat ccagcagcac ctgggccagc tgaatatgct gcacaggagg 300
agcggcctgc ccaggccatc cgacagcaac gccgtgtccc tggtcatgag aagaatcaga 360
aaggagaatg tggatgccgg cgagagagcc aagcaggccc tggcttttga gagaaccgat 420
ttcgatcagg tgaggtccct gatggagaat agcgacagat gccaggacat caggaatctg 480
gcctttctgg gcatcgccta caataccctg ctcaggatcg ccgagatcgc cagaatcaga 540
gtgaaggaca tcagcagaac cgacggcggc aggatgctga tccacatcgg cagaaccaag 600
accctggtga gcacagccgg cgtggagaag gccctgagcc tgggagtgac caagctggtg 660
gagaggtgga tctccgtgag cggcgtggcc gacgacccca ataactacct gttttgtagg 720
gtgaggaaga acggcgtggc cgcccccagc gctacaagcc aactgagcac cagagccctg 780
gagggcatct ttgaggccac acacaggctg atctacgggg ccaaggacga cagcggccag 840
agatacctgg cctggagcgg ccactccgcc agagtgggag ccgctagaga tatggccagg 900
gccggcgtga gcatccccga gatcatgcag gccggcggct ggaccaatgt gaatatcgtg 960
atgaactaca tcagaaacct ggattccgag acaggcgcta tggttagact gctggaggat 1020
ggc 1023
<210> 6
<211> 867
<212> DNA
<213> Ebola virus (Ebola virus)
<400> 6
atggaagctt catatgagag aggacgccca cgagctgcca gacagcattc aagggatgga 60
cacgaccacc atgttcgagc acgatcatca tccagagaga attatcgagg tgagtaccgt 120
caatcaagga gcgcctcaca agtgcgcgtt cctactgtat ttcataagaa gagagttgaa 180
ccattaacag ttcctccagc acctaaagac atatgtccga ccttgaaaaa aggatttttg 240
tgtgacagta gtttttgcaa aaaagatcac cagttggaga gtttaactga tagggaatta 300
ctcctactaa tcgcccgtaa gacttgtgga tcagtagaac aacaattaaa tataactgca 360
cccaaggact cgcgcttagc aaatccaacg gctgatgatt tccagcaaga ggaaggtcca 420
aaaattacct tgttgacact gatcaagacg gcagaacact gggcgagaca agacatcaga 480
accatagagg attcaaaatt aagagcattg ttgactctat gtgctgtgat gacgaggaaa 540
ttctcaaaat cccagctgag tcttttatgt gagacacacc taaggcgcga ggggcttggg 600
caagatcagg cagaacccgt tctcgaagta tatcaacgat tacacagtga taaaggaggc 660
agttttgaag ctgcactatg gcaacaatgg gaccgacaat ccctaattat gtttatcact 720
gcattcttga atattgctct ccagttaccg tgtgaaagtt ctgctgtcgt tgtttcaggg 780
ttaagaacat tggttcctca atcagataat gaggaagctt caaccaaccc cggcacatgc 840
tcatggtctg atgagggtac cccttaa 867
<210> 7
<211> 756
<212> DNA
<213> Ebola virus (Ebola virus)
<400> 7
atggctaaag ctacgggacg atacaatcta atatcgccca aaaaggacct ggagaaaggg 60
gttgtcttaa gcgacctctg taacttctta gttagccaaa ctattcaggg gtggaaggtt 120
tattgggctg gtattgagtt tgatgtgact cacaaaggaa tggccctatt gcatagactg 180
aaaactaatg actttgcccc tgcatggtca atgacaagga atctctttcc tcatttattt 240
caaaatccga attccacaat tgaatcaccg ctgtgggcat tgagagtcat ccttgcagca 300
gggatacagg accagctgat tgaccagtct ttgattgaac ccttagcagg agcccttggt 360
ctgatctctg attggctgct aacaaccaac actaaccatt tcaacatgcg aacacaacgt 420
gtcaaggaac aattgagcct aaaaatgctg tcgttgattc gatccaatat tctcaagttt 480
attaacaaat tggatgctct acatgtcgtg aactacaacg gattgttgag cagtattgaa 540
attggaactc aaaatcatac aatcatcata actcgaacta acatgggttt tctggtggag 600
ctccaagaac ccgacaaatc ggcaatgaac cgcatgaagc ctgggccggc gaaattttcc 660
ctccttcatg agtccacact gaaagcattt acacaaggga gctcgacacg aatgcaaagt 720
ttgattcttg aatttaatag ctctcttgct atctaa 756
<210> 8
<211> 6645
<212> DNA
<213> Ebola virus (Ebola virus)
<400> 8
atggctacac aacataccca atacccagac gctaggttat catcaccaat tgtattggac 60
caatgtgacc tagtcactcg agcttgcggg ttatattcat catactccct taatccgcaa 120
ctacgcaact gtaaactccc gaaacatatc taccgtttga aatacgatgt aactgttacc 180
aagttcttga gtgatgtacc agtggcgaca ttgcccatag atttcatagt cccagttctt 240
ctcaaggcac tgtcaggcaa tggattctgt cctgttgagc cgcggtgcca acagttctta 300
gatgaaatca ttaagtacac aatgcaagat gctctcttct tgaaatatta tctcaaaaat 360
gtgggtgctc aagaagactg tgttgatgaa cactttcaag agaaaatctt atcttcaatt 420
cagggcaatg aatttttaca tcaaatgttt ttctggtatg atctggctat tttaactcga 480
aggggtagat taaatcgagg aaactctaga tcaacatggt ttgttcatga tgatttaata 540
gacatcttag gctatgggga ctatgttttt tggaagatcc caatttcaat gttaccactg 600
aacacacaag gaatccccca tgctgctatg gactggtatc aggcatcagt attcaaagaa 660
gcggttcaag ggcatacaca cattgtttct gtttctactg ccgacgtgtt gataatgtgc 720
aaagatttaa ttacatgtcg attcaacaca actctaatct caaaaatagc agagattgag 780
gacccagttt gttctgatta tcccaatttt aagattgtgt ctatgcttta ccagagcgga 840
gattacttac tctccatatt agggtctgat gggtataaaa ttattaagtt cctcgaacca 900
ttgtgcttgg ccaaaattca attatgctca aagtacactg agaggaaggg ccgattctta 960
acacaaatgc atttagctgt aaatcacacc ctagaagaaa ttacagaaat gcgtgcacta 1020
aagccttcac aggctcaaaa gatccgtgag ttccatagaa cattgataag gctggagatg 1080
acgccacaac aactttgtga gctattttcc attcaaaaac actgggggca tcctgtgcta 1140
catagtgaaa cagcaatcca aaaagttaaa aaacatgcta cggtgctaaa agcattacgc 1200
cctatagtga ttttcgagac atactgtgtt tttaaatata gtattgccaa acattatttt 1260
gatagtcaag gatcttggta cagtgttact tcagatagga atctaacacc gggtcttaat 1320
tcttatatca aaagaaatca attccctccg ttgccaatga ttaaagaact actatgggaa 1380
ttttaccacc ttgaccaccc tccacttttc tcaaccaaaa ttattagtga cttaagtatt 1440
tttataaaag acagagctac cgcagtagaa aggacatgct gggatgcagt attcgagcct 1500
aatgttctag gatataatcc acctcacaaa tttagtacta aacgtgtacc ggaacaattt 1560
ttagagcaag aaaacttttc tattgagaat gttctttcct acgcacaaaa actggagtat 1620
ctactaccac aatatcggaa cttttctttc tcattgaaag agaaagagtt gaatgtaggt 1680
agaaccttcg gaaaattgcc ttatccgact cgcaatgttc aaacactttg tgaagctctg 1740
ttagctgatg gtcttgctaa agcatttcct agcaatatga tggtagttac ggaacgtgag 1800
caaaaagaaa gcttattgca tcaagcatca tggcaccaca caagtgatga ttttggtgaa 1860
catgccacag ttagagggag tagctttgta actgatttag agaaatacaa tcttgcattt 1920
agatatgagt ttacagcacc ttttatagaa tattgcaacc gttgctatgg tgttaagaat 1980
gtttttaatt ggatgcatta tacaatccca cagtgttata tgcatgtcag tgattattat 2040
aatccaccac ataacctcac actggagaat cgagacaacc cccccgaagg gcctagttca 2100
tacaggggtc atatgggagg gattgaagga ctgcaacaaa aactctggac aagtatttca 2160
tgtgctcaaa tttctttagt tgaaattaag actggtttta agttacgctc agctgtgatg 2220
ggtgacaatc agtgcattac tgttttatca gtcttcccct tagagactga cgcagacgag 2280
caggaacaga gcgccgaaga caatgcagcg agggtggccg ccagcctagc aaaagttaca 2340
agtgcctgtg gaatcttttt aaaacctgat gaaacatttg tacattcagg ttttatctat 2400
tttggaaaaa aacaatattt gaatggggtc caattgcctc agtcccttaa aacggctaca 2460
agaatggcac cattgtctga tgcaattttt gatgatcttc aagggaccct ggctagtata 2520
ggcactgctt ttgagcgatc catctctgag acacgacata tctttccttg caggataacc 2580
gcagctttcc atacgttttt ttcggtgaga atcttgcaat atcatcatct cgggttcaat 2640
aaaggttttg accttggaca gttgacactc ggcaaacctc tggatttcgg aacaatatca 2700
ttggcactag cggtcccgca ggtgcttgga gggttatcct tcttgaatcc tgagaaatgt 2760
ttctaccgga atctaggaga tccagttacc tcaggcttat tccagttaaa aacttatctc 2820
cgaatgattg agatggatga tttattctta cctttaattg cgaagaaccc tgggaactgc 2880
actgccattg actttgtgct aaatcctagc ggattaaatg tccctgggtc gcaagactta 2940
acttcatttc tgcgccagat tgtacgcagg accatcaccc taagtgcgaa aaacaaactt 3000
attaatacct tatttcatgc gtcagctgac ttcgaagacg aaatggtttg taaatggcta 3060
ttatcatcaa ctcctgttat gagtcgtttt gcggccgata tcttttcacg cacgccgagc 3120
gggaagcgat tgcaaattct aggatacctg gaaggaacac gcacattatt agcctctaag 3180
atcatcaaca ataatacaga gacaccggtt ttggacagac tgaggaaaat aacattgcaa 3240
aggtggagcc tatggtttag ttatcttgat cattgtgata atatcctggc ggaggcttta 3300
acccaaataa cttgcacagt tgatttagca cagattctga gggaatattc atgggctcat 3360
attttagagg gaagacctct tattggagcc acactcccat gtatgattga gcaattcaaa 3420
gtgttttggc tgaaacccta cgaacaatgt ccgcagtgtt caaatgcaaa gcaaccaggt 3480
gggaaaccat tcgtgtcagt ggcagtcaag aaacatattg ttagtgcatg gccgaacgca 3540
tcccgaataa gctggactat cggggatgga atcccataca ttggatcaag gacagaagat 3600
aagataggac aacctgctat taaaccaaaa tgtccctcag cagccttaag agaggccatt 3660
gaattggcgt cccgtttaac atgggtaact caaggcagtt cgaacagtga cttgctaata 3720
aaaccatttt tggaagcacg agtaaattta agtgttcaag aaatacttca aatgacccct 3780
tcacattact caggaaatat tgttcacagg tacaacgatc aatacagtcc tcattctttc 3840
atggccaatc gtatgagtaa ttcagcaacg cgattgattg tttctacaaa cactttaggt 3900
gagttttcag gaggtggcca gtctgcacgc gacagcaata ttattttcca gaatgttata 3960
aattatgcag ttgcactgtt cgatattaaa tttagaaaca ctgaggctac agatatccaa 4020
tataatcgtg ctcaccttca tctaactaag tgttgcaccc gcgaagtacc agctcagtat 4080
ttaacataca catctacatt ggatttagat ttaacaagat accgagaaaa cgaattgatt 4140
tatgacagta atcctctaaa aggaggactc aattgcaata tctcattcga taatccattt 4200
ttccaaggta aacggctgaa cattatagaa gatgatctta ttcgactgcc tcacttatct 4260
ggatgggagc tagccaagac catcatgcaa tcaattattt cagatagcaa caattcatct 4320
acagacccaa ttagcagtgg agaaacaaga tcattcacta cccatttctt aacttatccc 4380
aagataggac ttctgtacag ttttggggcc tttgtaagtt attatcttgg caatacaatt 4440
cttcggacta agaaattaac acttgacaat tttttatatt acttaactac tcaaattcat 4500
aatctaccac atcgctcatt gcgaatactt aagccaacat tcaaacatgc aagcgttatg 4560
tcacggttaa tgagtattga tcctcatttt tctatttaca taggcggtgc tgcaggtgac 4620
agaggactct cagatgcggc caggttattt ttgagaacgt ccatttcatc ttttcttaca 4680
tttgtaaaag aatggataat taatcgcgga acaattgtcc ctttatggat agtatatccg 4740
ctagagggtc aaaacccaac acctgtgaat aattttctct atcagatcgt agaactgctg 4800
gtgcatgatt catcaagaca acaggctttt aaaactacca taagtgatca tgtacatcct 4860
cacgacaatc ttgtttacac atgtaagagt acagccagca atttcttcca tgcatcattg 4920
gcgtactgga ggagcagaca cagaaacagc aaccgaaaat acttggcaag agactcttca 4980
actggatcaa gcacaaacaa cagtgatggt catattgaga gaagtcaaga acaaaccacc 5040
agagatccac atgatggcac tgaacggaat ctagtcctac aaatgagcca tgaaataaaa 5100
agaacgacaa ttccacaaga aaacacgcac cagggtccgt cgttccagtc ctttctaagt 5160
gactctgctt gtggtacagc aaatccaaaa ctaaatttcg ataggtcgag acacaatgtg 5220
aaatttcagg atcataactc ggcatccaag agggaaggtc atcaaataat ctcacaccgt 5280
ctagtcctac ctttctttac attatctcaa gggacacgcc aattaacgtc atccaatgag 5340
tcacaaaccc aagacgagat atcaaagtac ttacggcaat tgagatccgt cattgatacc 5400
acagtttatt gtagatttac cggtatagtc tcgtccatgc attacaaact tgatgaggtc 5460
ctttgggaaa tagagagttt caagtcggct gtgacgctag cagagggaga aggtgctggt 5520
gccttactat tgattcagaa ataccaagtt aagaccttat ttttcaacac gctagctact 5580
gagtccagta tagagtcaga aatagtatca ggaatgacta ctcctaggat gcttctacct 5640
gttatgtcaa aattccataa tgaccaaatt gagattattc ttaacaactc agcaagccaa 5700
ataacagaca taacaaatcc tacttggttt aaagaccaaa gagcaaggct acctaagcaa 5760
gtcgaggtta taaccatgga tgcagagaca acagagaata taaacagatc gaaattgtac 5820
gaagctgtat ataaattgat cttacaccat attgatccta gcgtattgaa agcagtggtc 5880
cttaaagtct ttctaagtga tactgagggt atgttatggc taaatgataa tttagccccg 5940
ttttttgcca ctggttattt aattaagcca ataacgtcaa gtgctagatc tagtgagtgg 6000
tatctttgtc tgacgaactt cttatcaact acacgtaaga tgccacacca aaaccatctc 6060
agttgtaaac aggtaatact tacggcattg caactgcaaa ttcaacgaag cccatactgg 6120
ctaagtcatt taactcagta tgctgactgt gagttacatt taagttatat ccgccttggt 6180
tttccatcat tagagaaagt actataccac aggtataacc tcgtcgattc aaaaagaggt 6240
ccactagtct ctatcactca gcacttagca catcttagag cagagattcg agaattaact 6300
aatgattata atcaacagcg acaaagtcgg actcaaacat atcactttat tcgtactgca 6360
aaaggacgaa tcacaaaact agtcaatgat tatttaaaat tctttcttat tgtgcaagca 6420
ttaaaacata atgggacatg gcaagctgag tttaagaaat taccagagtt gattagtgtg 6480
tgcaataggt tctaccatat tagagattgc aattgtgaag aacgtttctt agttcaaacc 6540
ttatatttac atagaatgca ggattctgaa gttaagctta tcgaaaggct gacagggctt 6600
ctgagtttat ttccggatgg tctctacagg tttgatggat cctga 6645
<210> 9
<211> 739
<212> PRT
<213> Ebola virus (Ebola virus)
<400> 9
Met Asp Ser Arg Pro Gln Lys Ile Trp Met Ala Pro Ser Leu Thr Glu
1 5 10 15
Ser Asp Met Asp Tyr His Lys Ile Leu Thr Ala Gly Leu Ser Val Gln
20 25 30
Gln Gly Ile Val Arg Gln Arg Val Ile Pro Val Tyr Gln Val Asn Asn
35 40 45
Leu Glu Glu Ile Cys Gln Leu Ile Ile Gln Ala Phe Glu Ala Gly Val
50 55 60
Asp Phe Gln Glu Ser Ala Asp Ser Phe Leu Leu Met Leu Cys Leu His
65 70 75 80
His Ala Tyr Gln Gly Asp Tyr Lys Leu Phe Leu Glu Ser Gly Ala Val
85 90 95
Lys Tyr Leu Glu Gly His Gly Phe Arg Phe Glu Val Lys Lys Arg Asp
100 105 110
Gly Val Lys Arg Leu Glu Glu Leu Leu Pro Ala Val Ser Ser Gly Lys
115 120 125
Asn Ile Lys Arg Thr Leu Ala Ala Met Pro Glu Glu Glu Thr Thr Glu
130 135 140
Ala Asn Ala Gly Gln Phe Leu Ser Phe Ala Ser Leu Phe Leu Pro Lys
145 150 155 160
Leu Val Val Gly Glu Lys Ala Cys Leu Glu Lys Val Gln Arg Gln Ile
165 170 175
Gln Val His Ala Glu Gln Gly Leu Ile Gln Tyr Pro Thr Ala Trp Gln
180 185 190
Ser Val Gly His Met Met Val Ile Phe Arg Leu Met Arg Thr Asn Phe
195 200 205
Leu Ile Lys Phe Leu Leu Ile His Gln Gly Met His Met Val Ala Gly
210 215 220
His Asp Ala Asn Asp Ala Val Ile Ser Asn Ser Val Ala Gln Ala Arg
225 230 235 240
Phe Ser Gly Leu Leu Ile Val Lys Thr Val Leu Asp His Ile Leu Gln
245 250 255
Lys Thr Glu Arg Gly Val Arg Leu His Pro Leu Ala Arg Thr Ala Lys
260 265 270
Val Lys Asn Glu Val Asn Ser Phe Lys Ala Ala Leu Ser Ser Leu Ala
275 280 285
Lys His Gly Glu Tyr Ala Pro Phe Ala Arg Leu Leu Asn Leu Ser Gly
290 295 300
Val Asn Asn Leu Glu His Gly Leu Phe Pro Gln Leu Ser Ala Ile Ala
305 310 315 320
Leu Gly Val Ala Thr Ala His Gly Ser Thr Leu Ala Gly Val Asn Val
325 330 335
Gly Glu Gln Tyr Gln Gln Leu Arg Glu Ala Ala Thr Glu Ala Glu Lys
340 345 350
Gln Leu Gln Gln Tyr Ala Glu Ser Arg Glu Leu Asp His Leu Gly Leu
355 360 365
Asp Asp Gln Glu Lys Lys Ile Leu Met Asn Phe His Gln Lys Lys Asn
370 375 380
Glu Ile Ser Phe Gln Gln Thr Asn Ala Met Val Thr Leu Arg Lys Glu
385 390 395 400
Arg Leu Ala Lys Leu Thr Glu Ala Ile Thr Ala Ala Ser Leu Pro Lys
405 410 415
Thr Ser Gly His Tyr Asp Asp Asp Asp Asp Ile Pro Phe Pro Gly Pro
420 425 430
Ile Asn Asp Asp Asp Asn Pro Gly His Gln Asp Asp Asp Pro Thr Asp
435 440 445
Ser Gln Asp Thr Thr Ile Pro Asp Val Val Val Asp Pro Asp Asp Gly
450 455 460
Ser Tyr Gly Glu Tyr Gln Ser Tyr Ser Glu Asn Gly Met Asn Ala Pro
465 470 475 480
Asp Asp Leu Val Leu Phe Asp Leu Asp Glu Asp Asp Glu Asp Thr Lys
485 490 495
Pro Val Pro Asn Arg Ser Thr Lys Gly Gly Gln Gln Lys Asn Ser Gln
500 505 510
Lys Gly Gln His Ile Glu Gly Arg Gln Thr Gln Ser Arg Pro Ile Gln
515 520 525
Asn Val Pro Gly Pro His Arg Thr Ile His His Ala Ser Ala Pro Leu
530 535 540
Thr Asp Asn Asp Arg Arg Asn Glu Pro Ser Gly Ser Thr Ser Pro Arg
545 550 555 560
Met Leu Thr Pro Ile Asn Glu Glu Ala Asp Pro Leu Asp Asp Ala Asp
565 570 575
Asp Glu Thr Ser Ser Leu Pro Pro Leu Glu Ser Asp Asp Glu Glu Gln
580 585 590
Asp Arg Asp Gly Thr Ser Asn Arg Thr Pro Thr Val Ala Pro Pro Ala
595 600 605
Pro Val Tyr Arg Asp His Ser Glu Lys Lys Glu Leu Pro Gln Asp Glu
610 615 620
Gln Gln Asp Gln Asp His Thr Gln Glu Ala Arg Asn Gln Asp Ser Asp
625 630 635 640
Asn Thr Gln Ser Glu His Ser Phe Glu Glu Met Tyr Arg His Ile Leu
645 650 655
Arg Ser Gln Gly Pro Phe Asp Ala Val Leu Tyr Tyr His Met Met Lys
660 665 670
Asp Glu Pro Val Val Phe Ser Thr Ser Asp Gly Lys Glu Tyr Thr Tyr
675 680 685
Pro Asp Ser Leu Glu Glu Glu Tyr Pro Pro Trp Leu Thr Glu Lys Glu
690 695 700
Ala Met Asn Glu Glu Asn Arg Phe Val Thr Leu Asp Gly Gln Gln Phe
705 710 715 720
Tyr Trp Pro Val Met Asn His Lys Asn Lys Phe Met Ala Ile Leu Gln
725 730 735
His His Gln
<210> 10
<211> 340
<212> PRT
<213> Ebola virus (Ebola virus)
<400> 10
Met Thr Thr Arg Thr Lys Gly Arg Gly His Thr Ala Ala Thr Thr Gln
1 5 10 15
Asn Asp Arg Met Pro Gly Pro Glu Leu Ser Gly Trp Ile Ser Glu Gln
20 25 30
Leu Met Thr Gly Arg Ile Pro Val Ser Asp Ile Phe Cys Asp Ile Glu
35 40 45
Asn Asn Pro Gly Leu Cys Tyr Ala Ser Gln Met Gln Gln Thr Lys Pro
50 55 60
Asn Pro Lys Thr Arg Asn Ser Gln Thr Gln Thr Asp Pro Ile Cys Asn
65 70 75 80
His Ser Phe Glu Glu Val Val Gln Thr Leu Ala Ser Leu Ala Thr Val
85 90 95
Val Gln Gln Gln Thr Ile Ala Ser Glu Ser Leu Glu Gln Arg Ile Thr
100 105 110
Ser Leu Glu Asn Gly Leu Lys Pro Val Tyr Asp Met Ala Lys Thr Ile
115 120 125
Ser Ser Leu Asn Arg Val Cys Ala Glu Met Val Ala Lys Tyr Asp Leu
130 135 140
Leu Val Met Thr Thr Gly Arg Ala Thr Ala Thr Ala Ala Ala Thr Glu
145 150 155 160
Ala Tyr Trp Ala Glu His Gly Gln Pro Pro Pro Gly Pro Ser Leu Tyr
165 170 175
Glu Glu Ser Ala Ile Arg Gly Lys Ile Glu Ser Arg Asp Glu Thr Val
180 185 190
Pro Gln Ser Val Arg Glu Ala Phe Asn Asn Leu Asn Ser Thr Thr Ser
195 200 205
Leu Thr Glu Glu Asn Phe Gly Lys Pro Asp Ile Ser Ala Lys Asp Leu
210 215 220
Arg Asn Ile Met Tyr Asp His Leu Pro Gly Phe Gly Thr Ala Phe His
225 230 235 240
Gln Leu Val Gln Val Ile Cys Lys Leu Gly Lys Asp Ser Asn Ser Leu
245 250 255
Asp Ile Ile His Ala Glu Phe Gln Ala Ser Leu Ala Glu Gly Asp Ser
260 265 270
Pro Gln Cys Ala Leu Ile Gln Ile Thr Lys Arg Val Pro Ile Phe Gln
275 280 285
Asp Ala Ala Pro Pro Val Ile His Ile Arg Ser Arg Gly Asp Ile Pro
290 295 300
Arg Ala Cys Gln Lys Ser Leu Arg Pro Val Pro Pro Ser Pro Lys Ile
305 310 315 320
Asp Arg Gly Trp Val Cys Val Phe Gln Leu Gln Asp Gly Lys Thr Leu
325 330 335
Gly Leu Lys Ile
340
<210> 11
<211> 326
<212> PRT
<213> Ebola virus (Ebola virus)
<400> 11
Met Arg Arg Val Ile Leu Pro Thr Ala Pro Pro Glu Tyr Met Glu Ala
1 5 10 15
Ile Tyr Pro Val Arg Ser Asn Ser Thr Ile Ala Arg Gly Gly Asn Ser
20 25 30
Asn Thr Gly Phe Leu Thr Pro Glu Ser Val Asn Gly Asp Thr Pro Ser
35 40 45
Asn Pro Leu Arg Pro Ile Ala Asp Asp Thr Ile Asp His Ala Ser His
50 55 60
Thr Pro Gly Ser Val Ser Ser Ala Phe Ile Leu Glu Ala Met Val Asn
65 70 75 80
Val Ile Ser Gly Pro Lys Val Leu Met Lys Gln Ile Pro Ile Trp Leu
85 90 95
Pro Leu Gly Val Ala Asp Gln Lys Thr Tyr Ser Phe Asp Ser Thr Thr
100 105 110
Ala Ala Ile Met Leu Ala Ser Tyr Thr Ile Thr His Phe Gly Lys Ala
115 120 125
Thr Asn Pro Leu Val Arg Val Asn Arg Leu Gly Pro Gly Ile Pro Asp
130 135 140
His Pro Leu Arg Leu Leu Arg Ile Gly Asn Gln Ala Phe Leu Gln Glu
145 150 155 160
Phe Val Leu Pro Pro Val Gln Leu Pro Gln Tyr Phe Thr Phe Asp Leu
165 170 175
Thr Ala Leu Lys Leu Ile Thr Gln Pro Leu Pro Ala Ala Thr Trp Thr
180 185 190
Asp Asp Thr Pro Thr Gly Ser Asn Gly Ala Leu Arg Pro Gly Ile Ser
195 200 205
Phe His Pro Lys Leu Arg Pro Ile Leu Leu Pro Asn Lys Ser Gly Lys
210 215 220
Lys Gly Asn Ser Ala Asp Leu Thr Ser Pro Glu Lys Ile Gln Ala Ile
225 230 235 240
Met Thr Ser Leu Gln Asp Phe Lys Ile Val Pro Ile Asp Pro Thr Lys
245 250 255
Asn Ile Met Gly Ile Glu Val Pro Glu Thr Leu Val His Lys Leu Thr
260 265 270
Gly Lys Lys Val Thr Ser Lys Asn Gly Gln Pro Ile Ile Pro Val Leu
275 280 285
Leu Pro Lys Tyr Ile Gly Leu Asp Pro Val Ala Pro Gly Asp Leu Thr
290 295 300
Met Val Ile Thr Gln Asp Cys Asp Thr Cys His Ser Pro Ala Ser Leu
305 310 315 320
Pro Ala Val Ile Glu Lys
325
<210> 12
<211> 288
<212> PRT
<213> Ebola virus (Ebola virus)
<400> 12
Met Glu Ala Ser Tyr Glu Arg Gly Arg Pro Arg Ala Ala Arg Gln His
1 5 10 15
Ser Arg Asp Gly His Asp His His Val Arg Ala Arg Ser Ser Ser Arg
20 25 30
Glu Asn Tyr Arg Gly Glu Tyr Arg Gln Ser Arg Ser Ala Ser Gln Val
35 40 45
Arg Val Pro Thr Val Phe His Lys Lys Arg Val Glu Pro Leu Thr Val
50 55 60
Pro Pro Ala Pro Lys Asp Ile Cys Pro Thr Leu Lys Lys Gly Phe Leu
65 70 75 80
Cys Asp Ser Ser Phe Cys Lys Lys Asp His Gln Leu Glu Ser Leu Thr
85 90 95
Asp Arg Glu Leu Leu Leu Leu Ile Ala Arg Lys Thr Cys Gly Ser Val
100 105 110
Glu Gln Gln Leu Asn Ile Thr Ala Pro Lys Asp Ser Arg Leu Ala Asn
115 120 125
Pro Thr Ala Asp Asp Phe Gln Gln Glu Glu Gly Pro Lys Ile Thr Leu
130 135 140
Leu Thr Leu Ile Lys Thr Ala Glu His Trp Ala Arg Gln Asp Ile Arg
145 150 155 160
Thr Ile Glu Asp Ser Lys Leu Arg Ala Leu Leu Thr Leu Cys Ala Val
165 170 175
Met Thr Arg Lys Phe Ser Lys Ser Gln Leu Ser Leu Leu Cys Glu Thr
180 185 190
His Leu Arg Arg Glu Gly Leu Gly Gln Asp Gln Ala Glu Pro Val Leu
195 200 205
Glu Val Tyr Gln Arg Leu His Ser Asp Lys Gly Gly Ser Phe Glu Ala
210 215 220
Ala Leu Trp Gln Gln Trp Asp Arg Gln Ser Leu Ile Met Phe Ile Thr
225 230 235 240
Ala Phe Leu Asn Ile Ala Leu Gln Leu Pro Cys Glu Ser Ser Ala Val
245 250 255
Val Val Ser Gly Leu Arg Thr Leu Val Pro Gln Ser Asp Asn Glu Glu
260 265 270
Ala Ser Thr Asn Pro Gly Thr Cys Ser Trp Ser Asp Glu Gly Thr Pro
275 280 285
<210> 13
<211> 251
<212> PRT
<213> Ebola virus (Ebola virus)
<400> 13
Met Ala Lys Ala Thr Gly Arg Tyr Asn Leu Ile Ser Pro Lys Lys Asp
1 5 10 15
Leu Glu Lys Gly Val Val Leu Ser Asp Leu Cys Asn Phe Leu Val Ser
20 25 30
Gln Thr Ile Gln Gly Trp Lys Val Tyr Trp Ala Gly Ile Glu Phe Asp
35 40 45
Val Thr His Lys Gly Met Ala Leu Leu His Arg Leu Lys Thr Asn Asp
50 55 60
Phe Ala Pro Ala Trp Ser Met Thr Arg Asn Leu Phe Pro His Leu Phe
65 70 75 80
Gln Asn Pro Asn Ser Thr Ile Glu Ser Pro Leu Trp Ala Leu Arg Val
85 90 95
Ile Leu Ala Ala Gly Ile Gln Asp Gln Leu Ile Asp Gln Ser Leu Ile
100 105 110
Glu Pro Leu Ala Gly Ala Leu Gly Leu Ile Ser Asp Trp Leu Leu Thr
115 120 125
Thr Asn Thr Asn His Phe Asn Met Arg Thr Gln Arg Val Lys Glu Gln
130 135 140
Leu Ser Leu Lys Met Leu Ser Leu Ile Arg Ser Asn Ile Leu Lys Phe
145 150 155 160
Ile Asn Lys Leu Asp Ala Leu His Val Val Asn Tyr Asn Gly Leu Leu
165 170 175
Ser Ser Ile Glu Ile Gly Thr Gln Asn His Thr Ile Ile Ile Thr Arg
180 185 190
Thr Asn Met Gly Phe Leu Val Glu Leu Gln Glu Pro Asp Lys Ser Ala
195 200 205
Met Asn Arg Met Lys Pro Gly Pro Ala Lys Phe Ser Leu Leu His Glu
210 215 220
Ser Thr Leu Lys Ala Phe Thr Gln Gly Ser Ser Thr Arg Met Gln Ser
225 230 235 240
Leu Ile Leu Glu Phe Asn Ser Ser Leu Ala Ile
245 250
<210> 14
<211> 2214
<212> PRT
<213> Ebola virus (Ebola virus)
<400> 14
Met Ala Thr Gln His Thr Gln Tyr Pro Asp Ala Arg Leu Ser Ser Pro
1 5 10 15
Ile Val Leu Asp Gln Cys Asp Leu Val Thr Arg Ala Cys Gly Leu Tyr
20 25 30
Ser Ser Tyr Ser Leu Asn Pro Gln Leu Arg Asn Cys Lys Leu Pro Lys
35 40 45
His Ile Tyr Arg Leu Lys Tyr Asp Val Thr Val Thr Lys Phe Leu Ser
50 55 60
Asp Val Pro Val Ala Thr Leu Pro Ile Asp Phe Ile Val Pro Val Leu
65 70 75 80
Leu Lys Ala Leu Ser Gly Asn Gly Phe Cys Pro Val Glu Pro Arg Cys
85 90 95
Gln Gln Phe Leu Asp Glu Ile Ile Lys Tyr Thr Met Gln Asp Ala Leu
100 105 110
Phe Leu Lys Tyr Tyr Leu Lys Asn Val Gly Ala Gln Glu Asp Cys Val
115 120 125
Asp Glu His Phe Gln Glu Lys Ile Leu Ser Ser Ile Gln Gly Asn Glu
130 135 140
Phe Leu His Gln Met Phe Phe Trp Tyr Asp Leu Ala Ile Leu Thr Arg
145 150 155 160
Arg Gly Arg Leu Asn Arg Gly Asn Ser Arg Ser Thr Trp Phe Val His
165 170 175
Asp Asp Leu Ile Asp Ile Leu Gly Tyr Gly Asp Tyr Val Phe Trp Lys
180 185 190
Ile Pro Ile Ser Met Leu Pro Leu Asn Thr Gln Gly Ile Pro His Ala
195 200 205
Ala Met Asp Trp Tyr Gln Ala Ser Val Phe Lys Glu Ala Val Gln Gly
210 215 220
His Thr His Ile Val Ser Val Ser Thr Ala Asp Val Leu Ile Met Cys
225 230 235 240
Lys Asp Leu Ile Thr Cys Arg Phe Asn Thr Thr Leu Ile Ser Lys Ile
245 250 255
Ala Glu Ile Glu Asp Pro Val Cys Ser Asp Tyr Pro Asn Phe Lys Ile
260 265 270
Val Ser Met Leu Tyr Gln Ser Gly Asp Tyr Leu Leu Ser Ile Leu Gly
275 280 285
Ser Asp Gly Tyr Lys Ile Ile Lys Phe Leu Glu Pro Leu Cys Leu Ala
290 295 300
Lys Ile Gln Leu Cys Ser Lys Tyr Thr Glu Arg Lys Gly Arg Phe Leu
305 310 315 320
Thr Gln Met His Leu Ala Val Asn His Thr Leu Glu Glu Ile Thr Glu
325 330 335
Met Arg Ala Leu Lys Pro Ser Gln Ala Gln Lys Ile Arg Glu Phe His
340 345 350
Arg Thr Leu Ile Arg Leu Glu Met Thr Pro Gln Gln Leu Cys Glu Leu
355 360 365
Phe Ser Ile Gln Lys His Trp Gly His Pro Val Leu His Ser Glu Thr
370 375 380
Ala Ile Gln Lys Val Lys Lys His Ala Thr Val Leu Lys Ala Leu Arg
385 390 395 400
Pro Ile Val Ile Phe Glu Thr Tyr Cys Val Phe Lys Tyr Ser Ile Ala
405 410 415
Lys His Tyr Phe Asp Ser Gln Gly Ser Trp Tyr Ser Val Thr Ser Asp
420 425 430
Arg Asn Leu Thr Pro Gly Leu Asn Ser Tyr Ile Lys Arg Asn Gln Phe
435 440 445
Pro Pro Leu Pro Met Ile Lys Glu Leu Leu Trp Glu Phe Tyr His Leu
450 455 460
Asp His Pro Pro Leu Phe Ser Thr Lys Ile Ile Ser Asp Leu Ser Ile
465 470 475 480
Phe Ile Lys Asp Arg Ala Thr Ala Val Glu Arg Thr Cys Trp Asp Ala
485 490 495
Val Phe Glu Pro Asn Val Leu Gly Tyr Asn Pro Pro His Lys Phe Ser
500 505 510
Thr Lys Arg Val Pro Glu Gln Phe Leu Glu Gln Glu Asn Phe Ser Ile
515 520 525
Glu Asn Val Leu Ser Tyr Ala Gln Lys Leu Glu Tyr Leu Leu Pro Gln
530 535 540
Tyr Arg Asn Phe Ser Phe Ser Leu Lys Glu Lys Glu Leu Asn Val Gly
545 550 555 560
Arg Thr Phe Gly Lys Leu Pro Tyr Pro Thr Arg Asn Val Gln Thr Leu
565 570 575
Cys Glu Ala Leu Leu Ala Asp Gly Leu Ala Lys Ala Phe Pro Ser Asn
580 585 590
Met Met Val Val Thr Glu Arg Glu Gln Lys Glu Ser Leu Leu His Gln
595 600 605
Ala Ser Trp His His Thr Ser Asp Asp Phe Gly Glu His Ala Thr Val
610 615 620
Arg Gly Ser Ser Phe Val Thr Asp Leu Glu Lys Tyr Asn Leu Ala Phe
625 630 635 640
Arg Tyr Glu Phe Thr Ala Pro Phe Ile Glu Tyr Cys Asn Arg Cys Tyr
645 650 655
Gly Val Lys Asn Val Phe Asn Trp Met His Tyr Thr Ile Pro Gln Cys
660 665 670
Tyr Met His Val Ser Asp Tyr Tyr Asn Pro Pro His Asn Leu Thr Leu
675 680 685
Glu Asn Arg Asp Asn Pro Pro Glu Gly Pro Ser Ser Tyr Arg Gly His
690 695 700
Met Gly Gly Ile Glu Gly Leu Gln Gln Lys Leu Trp Thr Ser Ile Ser
705 710 715 720
Cys Ala Gln Ile Ser Leu Val Glu Ile Lys Thr Gly Phe Lys Leu Arg
725 730 735
Ser Ala Val Met Gly Asp Asn Gln Cys Ile Thr Val Leu Ser Val Phe
740 745 750
Pro Leu Glu Thr Asp Ala Asp Glu Gln Glu Gln Ser Ala Glu Asp Asn
755 760 765
Ala Ala Arg Val Ala Ala Ser Leu Ala Lys Val Thr Ser Ala Cys Gly
770 775 780
Ile Phe Leu Lys Pro Asp Glu Thr Phe Val His Ser Gly Phe Ile Tyr
785 790 795 800
Phe Gly Lys Lys Gln Tyr Leu Asn Gly Val Gln Leu Pro Gln Ser Leu
805 810 815
Lys Thr Ala Thr Arg Met Ala Pro Leu Ser Asp Ala Ile Phe Asp Asp
820 825 830
Leu Gln Gly Thr Leu Ala Ser Ile Gly Thr Ala Phe Glu Arg Ser Ile
835 840 845
Ser Glu Thr Arg His Ile Phe Pro Cys Arg Ile Thr Ala Ala Phe His
850 855 860
Thr Phe Phe Ser Val Arg Ile Leu Gln Tyr His His Leu Gly Phe Asn
865 870 875 880
Lys Gly Phe Asp Leu Gly Gln Leu Thr Leu Gly Lys Pro Leu Asp Phe
885 890 895
Gly Thr Ile Ser Leu Ala Leu Ala Val Pro Gln Val Leu Gly Gly Leu
900 905 910
Ser Phe Leu Asn Pro Glu Lys Cys Phe Tyr Arg Asn Leu Gly Asp Pro
915 920 925
Val Thr Ser Gly Leu Phe Gln Leu Lys Thr Tyr Leu Arg Met Ile Glu
930 935 940
Met Asp Asp Leu Phe Leu Pro Leu Ile Ala Lys Asn Pro Gly Asn Cys
945 950 955 960
Thr Ala Ile Asp Phe Val Leu Asn Pro Ser Gly Leu Asn Val Pro Gly
965 970 975
Ser Gln Asp Leu Thr Ser Phe Leu Arg Gln Ile Val Arg Arg Thr Ile
980 985 990
Thr Leu Ser Ala Lys Asn Lys Leu Ile Asn Thr Leu Phe His Ala Ser
995 1000 1005
Ala Asp Phe Glu Asp Glu Met Val Cys Lys Trp Leu Leu Ser Ser
1010 1015 1020
Thr Pro Val Met Ser Arg Phe Ala Ala Asp Ile Phe Ser Arg Thr
1025 1030 1035
Pro Ser Gly Lys Arg Leu Gln Ile Leu Gly Tyr Leu Glu Gly Thr
1040 1045 1050
Arg Thr Leu Leu Ala Ser Lys Ile Ile Asn Asn Asn Thr Glu Thr
1055 1060 1065
Pro Val Leu Asp Arg Leu Arg Lys Ile Thr Leu Gln Arg Trp Ser
1070 1075 1080
Leu Trp Phe Ser Tyr Leu Asp His Cys Asp Asn Ile Leu Ala Glu
1085 1090 1095
Ala Leu Thr Gln Ile Thr Cys Thr Val Asp Leu Ala Gln Ile Leu
1100 1105 1110
Arg Glu Tyr Ser Trp Ala His Ile Leu Glu Gly Arg Pro Leu Ile
1115 1120 1125
Gly Ala Thr Leu Pro Cys Met Ile Glu Gln Phe Lys Val Phe Trp
1130 1135 1140
Leu Lys Pro Tyr Glu Gln Cys Pro Gln Cys Ser Asn Ala Lys Gln
1145 1150 1155
Pro Gly Gly Lys Pro Phe Val Ser Val Ala Val Lys Lys His Ile
1160 1165 1170
Val Ser Ala Trp Pro Asn Ala Ser Arg Ile Ser Trp Thr Ile Gly
1175 1180 1185
Asp Gly Ile Pro Tyr Ile Gly Ser Arg Thr Glu Asp Lys Ile Gly
1190 1195 1200
Gln Pro Ala Ile Lys Pro Lys Cys Pro Ser Ala Ala Leu Arg Glu
1205 1210 1215
Ala Ile Glu Leu Ala Ser Arg Leu Thr Trp Val Thr Gln Gly Ser
1220 1225 1230
Ser Asn Ser Asp Leu Leu Ile Lys Pro Phe Leu Glu Ala Arg Val
1235 1240 1245
Asn Leu Ser Val Gln Glu Ile Leu Gln Met Thr Pro Ser His Tyr
1250 1255 1260
Ser Gly Asn Ile Val His Arg Tyr Asn Asp Gln Tyr Ser Pro His
1265 1270 1275
Ser Phe Met Ala Asn Arg Met Ser Asn Ser Ala Thr Arg Leu Ile
1280 1285 1290
Val Ser Thr Asn Thr Leu Gly Glu Phe Ser Gly Gly Gly Gln Ser
1295 1300 1305
Ala Arg Asp Ser Asn Ile Ile Phe Gln Asn Val Ile Asn Tyr Ala
1310 1315 1320
Val Ala Leu Phe Asp Ile Lys Phe Arg Asn Thr Glu Ala Thr Asp
1325 1330 1335
Ile Gln Tyr Asn Arg Ala His Leu His Leu Thr Lys Cys Cys Thr
1340 1345 1350
Arg Glu Val Pro Ala Gln Tyr Leu Thr Tyr Thr Ser Thr Leu Asp
1355 1360 1365
Leu Asp Leu Thr Arg Tyr Arg Glu Asn Glu Leu Ile Tyr Asp Ser
1370 1375 1380
Asn Pro Leu Lys Gly Gly Leu Asn Cys Asn Ile Ser Phe Asp Asn
1385 1390 1395
Pro Phe Phe Gln Gly Lys Arg Leu Asn Ile Ile Glu Asp Asp Leu
1400 1405 1410
Ile Arg Leu Pro His Leu Ser Gly Trp Glu Leu Ala Lys Thr Ile
1415 1420 1425
Met Gln Ser Ile Ile Ser Asp Ser Asn Asn Ser Ser Thr Asp Pro
1430 1435 1440
Ile Ser Ser Gly Glu Thr Arg Ser Phe Thr Thr His Phe Leu Thr
1445 1450 1455
Tyr Pro Lys Ile Gly Leu Leu Tyr Ser Phe Gly Ala Phe Val Ser
1460 1465 1470
Tyr Tyr Leu Gly Asn Thr Ile Leu Arg Thr Lys Lys Leu Thr Leu
1475 1480 1485
Asp Asn Phe Leu Tyr Tyr Leu Thr Thr Gln Ile His Asn Leu Pro
1490 1495 1500
His Arg Ser Leu Arg Ile Leu Lys Pro Thr Phe Lys His Ala Ser
1505 1510 1515
Val Met Ser Arg Leu Met Ser Ile Asp Pro His Phe Ser Ile Tyr
1520 1525 1530
Ile Gly Gly Ala Ala Gly Asp Arg Gly Leu Ser Asp Ala Ala Arg
1535 1540 1545
Leu Phe Leu Arg Thr Ser Ile Ser Ser Phe Leu Thr Phe Val Lys
1550 1555 1560
Glu Trp Ile Ile Asn Arg Gly Thr Ile Val Pro Leu Trp Ile Val
1565 1570 1575
Tyr Pro Leu Glu Gly Gln Asn Pro Thr Pro Val Asn Asn Phe Leu
1580 1585 1590
Tyr Gln Ile Val Glu Leu Leu Val His Asp Ser Ser Arg Gln Gln
1595 1600 1605
Ala Phe Lys Thr Thr Ile Ser Asp His Val His Pro His Asp Asn
1610 1615 1620
Leu Val Tyr Thr Cys Lys Ser Thr Ala Ser Asn Phe Phe His Ala
1625 1630 1635
Ser Leu Ala Tyr Trp Arg Ser Arg His Arg Asn Ser Asn Arg Lys
1640 1645 1650
Tyr Leu Ala Arg Asp Ser Ser Thr Gly Ser Ser Thr Asn Asn Ser
1655 1660 1665
Asp Gly His Ile Glu Arg Ser Gln Glu Gln Thr Thr Arg Asp Pro
1670 1675 1680
His Asp Gly Thr Glu Arg Asn Leu Val Leu Gln Met Ser His Glu
1685 1690 1695
Ile Lys Arg Thr Thr Ile Pro Gln Glu Asn Thr His Gln Gly Pro
1700 1705 1710
Ser Phe Gln Ser Phe Leu Ser Asp Ser Ala Cys Gly Thr Ala Asn
1715 1720 1725
Pro Lys Leu Asn Phe Asp Arg Ser Arg His Asn Val Lys Phe Gln
1730 1735 1740
Asp His Asn Ser Ala Ser Lys Arg Glu Gly His Gln Ile Ile Ser
1745 1750 1755
His Arg Leu Val Leu Pro Phe Phe Thr Leu Ser Gln Gly Thr Arg
1760 1765 1770
Gln Leu Thr Ser Ser Asn Glu Ser Gln Thr Gln Asp Glu Ile Ser
1775 1780 1785
Lys Tyr Leu Arg Gln Leu Arg Ser Val Ile Asp Thr Thr Val Tyr
1790 1795 1800
Cys Arg Phe Thr Gly Ile Val Ser Ser Met His Tyr Lys Leu Asp
1805 1810 1815
Glu Val Leu Trp Glu Ile Glu Ser Phe Lys Ser Ala Val Thr Leu
1820 1825 1830
Ala Glu Gly Glu Gly Ala Gly Ala Leu Leu Leu Ile Gln Lys Tyr
1835 1840 1845
Gln Val Lys Thr Leu Phe Phe Asn Thr Leu Ala Thr Glu Ser Ser
1850 1855 1860
Ile Glu Ser Glu Ile Val Ser Gly Met Thr Thr Pro Arg Met Leu
1865 1870 1875
Leu Pro Val Met Ser Lys Phe His Asn Asp Gln Ile Glu Ile Ile
1880 1885 1890
Leu Asn Asn Ser Ala Ser Gln Ile Thr Asp Ile Thr Asn Pro Thr
1895 1900 1905
Trp Phe Lys Asp Gln Arg Ala Arg Leu Pro Lys Gln Val Glu Val
1910 1915 1920
Ile Thr Met Asp Ala Glu Thr Thr Glu Asn Ile Asn Arg Ser Lys
1925 1930 1935
Leu Tyr Glu Ala Val Tyr Lys Leu Ile Leu His His Ile Asp Pro
1940 1945 1950
Ser Val Leu Lys Ala Val Val Leu Lys Val Phe Leu Ser Asp Thr
1955 1960 1965
Glu Gly Met Leu Trp Leu Asn Asp Asn Leu Ala Pro Phe Phe Ala
1970 1975 1980
Thr Gly Tyr Leu Ile Lys Pro Ile Thr Ser Ser Ala Arg Ser Ser
1985 1990 1995
Glu Trp Tyr Leu Cys Leu Thr Asn Phe Leu Ser Thr Thr Arg Lys
2000 2005 2010
Met Pro His Gln Asn His Leu Ser Cys Lys Gln Val Ile Leu Thr
2015 2020 2025
Ala Leu Gln Leu Gln Ile Gln Arg Ser Pro Tyr Trp Leu Ser His
2030 2035 2040
Leu Thr Gln Tyr Ala Asp Cys Glu Leu His Leu Ser Tyr Ile Arg
2045 2050 2055
Leu Gly Phe Pro Ser Leu Glu Lys Val Leu Tyr His Arg Tyr Asn
2060 2065 2070
Leu Val Asp Ser Lys Arg Gly Pro Leu Val Ser Ile Thr Gln His
2075 2080 2085
Leu Ala His Leu Arg Ala Glu Ile Arg Glu Leu Thr Asn Asp Tyr
2090 2095 2100
Asn Gln Gln Arg Gln Ser Arg Thr Gln Thr Tyr His Phe Ile Arg
2105 2110 2115
Thr Ala Lys Gly Arg Ile Thr Lys Leu Val Asn Asp Tyr Leu Lys
2120 2125 2130
Phe Phe Leu Ile Val Gln Ala Leu Lys His Asn Gly Thr Trp Gln
2135 2140 2145
Ala Glu Phe Lys Lys Leu Pro Glu Leu Ile Ser Val Cys Asn Arg
2150 2155 2160
Phe Tyr His Ile Arg Asp Cys Asn Cys Glu Glu Arg Phe Leu Val
2165 2170 2175
Gln Thr Leu Tyr Leu His Arg Met Gln Asp Ser Glu Val Lys Leu
2180 2185 2190
Ile Glu Arg Leu Thr Gly Leu Leu Ser Leu Phe Pro Asp Gly Leu
2195 2200 2205
Tyr Arg Phe Asp Gly Ser
2210
<210> 15
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> loxP site sequence recognized by Cre recombinase
<220>
<221> misc_feature
<222> (14)..(16)
<223> n=a or T or C or G
<220>
<221> misc_feature
<222> (19)..(21)
<223> n=a or T or C or G
<400> 15
ataacttcgt atannntann ntatacgaag ttat 34

Claims (10)

1. A defective recombinant filovirus, characterized in that the coding sequence of one or more key viral proteins in the genome of said defective recombinant filovirus is replaced by a recombinase coding sequence,
wherein the key viral protein is selected from the group consisting of: GP protein, VP40 protein, VP24 protein, or a combination thereof; the recombinase is selected from the group consisting of: cre recombinase, flp recombinase, dre recombinase, or a combination thereof.
2. An isolated polynucleotide encoding the defective recombinant filovirus of claim 1.
3. A vector comprising the polynucleotide of claim 2.
4. A host cell comprising the vector of claim 3, or having integrated into its genome the polynucleotide of claim 2.
5. A composition, wherein the composition comprises:
(a) The vector of claim 3, wherein the coding sequence for a key viral protein in the vector is replaced with a recombinase coding sequence; and
(b) A regulated expression vector, wherein said regulated expression vector is regulated by expression of said recombinase of (a), and expresses said critical viral protein of (a) under suitable conditions.
6. A method for preparing a defective recombinant filovirus, the method comprising the steps of:
(a) Providing a vector according to claim 3, wherein the coding sequence for a key viral protein in the vector is replaced with a recombinase coding sequence;
(b) Constructing a regulatory expression vector, wherein said regulatory expression vector is regulated by expression of said recombinase of (a), and expressing said critical viral protein of (a) under appropriate conditions;
(c) Transduction of the regulated expression vector of step (b) into a eukaryotic cell, thereby obtaining a complementing cell;
(d) Optionally, constructing other viral protein expression vectors; and
(e) Transduction of the vector of step (a) and optionally other viral protein expression vectors into the complementary cells obtained in step (c), thereby obtaining the defective recombinant filovirus.
7. Use of a composition according to claim 5 for the preparation of a defective recombinant filovirus according to claim 1.
8. A pharmaceutical composition, the pharmaceutical composition comprising:
(i) An inactivated or attenuated defective recombinant filovirus according to claim 1; and
(ii) A pharmaceutically acceptable carrier, diluent or excipient.
9. A test agent comprising the defective recombinant filovirus of claim 1, wherein the defective recombinant filovirus is conjugated with a detectable label.
10. A method for in vitro detection of filovirus antibodies in a sample, said method comprising the steps of:
(1) Contacting the sample with the defective recombinant filovirus of claim 1 in vitro; and
(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of the filovirus antibody in the sample.
CN202210754314.5A 2022-06-28 2022-06-28 Preparation method and application of defective filovirus Pending CN117343911A (en)

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CN103993012B (en) * 2007-12-27 2023-01-10 苏黎士大学 Replication-defective arenavirus vectors
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