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Patent Analysis of

Construction of West Nile virus and dengue virus chimeras for use in a live virus vaccine to prevent disease caused by West Nile virus

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10058602

Application Number

US14/305572

Application Date

16 June 2014

Publication Date

28 August 2018

Current Assignee

THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES,THE GOVERNMENT OF THE UNITED STATES, AS REPRESENTED BY THE SECRETARY OF THE ARMY

Original Assignee (Applicant)

THE U.S.A, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH & HUMAN SERVICES,THE WALTER REED ARMY INSTITUTE OF RESEARCH

International Classification

A61K45/06,C07K14/005,A61K39/12,C12N7/00,C12N15/86

Cooperative Classification

A61K39/12,C07K14/005,C12N7/00,C12N15/86,C12Q1/6888

Inventor

PLETNEV, ALEXANDER G.,PUTNAK, JOSEPH R.,CHANOCK, ROBERT M.,MURPHY, BRIAN R.,WHITEHEAD, STEPHEN S.,BLANEY, JOSEPH E.

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10058602 Construction West Nile virus 1 US10058602 Construction West Nile virus 2 US10058602 Construction West Nile virus 3
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Abstract

The present invention relates to attenuated, immunogenic West Nile virus chimeras built on a dengue virus backbone for the production of immunogenic, live, attenuated West Nile virus vaccines.

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Claims

1. A nucleic acid chimera comprising a first nucleotide sequence encoding two structural proteins from a West Nile virus, wherein the structural proteins are premembrane/membrane (prM) and envelope (E), and a second nucleotide sequence encoding capsid (C) and nonstructural proteins from a wild-type strain of dengue virus, wherein the dengue virus is attenuated by a deletion of about 30 nucleotides from the 3′ untranslated region of the dengue genome corresponding to the TL2 stem-loop structure.

2. The nucleic acid chimera of claim 1, wherein the dengue virus is selected from the group consisting of dengue type 1 virus, dengue type 2 virus, dengue type 3 virus, and dengue type 4 virus.

3. The nucleic acid chimera of claim 1, wherein the dengue virus is adapted for increased growth in Vero cells.

4. The nucleic acid chimera of claim 3, wherein the dengue virus is dengue type 4 virus and the deletion of about 30 nucleotides from the 3′ untranslated region corresponds to nucleotides 10478-10507.

5. The nucleic acid chimera of claim 3, wherein the dengue virus is dengue type 1 virus and the deletion of about 30 nucleotides from the 3′ untranslated region corresponds to nucleotides 10562-10591.

6. The nucleic acid chimera of claim 3, wherein the dengue virus is dengue type 2 virus and the deletion of about 30nucleotides from the 3′ untranslated region corresponds to nucleotides 10541-10570.

7. The nucleic acid chimera of claim 3, wherein the dengue virus is dengue type 3 virus and the deletion of about 30nucleotides from the 3′ untranslated region corresponds to nucleotides 10535-10565.

8. The nucleic acid chimera of claim 1, wherein the dengue virus is dengue type 4 virus, wherein a cleavage site is utilized for joining a dengue virus capsid protein and a West Nile virus prM protein, and wherein the West Nile virus prM protein contains aspartic acid (Asp) at a position 3 amino acids downstream of the cleavage site and contains threonine (Thr) at a position 6 amino acids downstream of the cleavage site.

9. A virus chimera comprising at least one of the nucleic acid chimera of claim 1.

10. An immunogenic composition comprising the nucleic acid chimera of claim 1 and a pharmaceutically acceptable carrier.

11. A method of inducing an immune response in a subject comprising administering an effective amount of the composition of claim 10 to the subject.

12. The method of claim 11, wherein the subject is selected from the group consisting of a non-human primate, a human, a horse, and a bird.

13. A vaccine composition comprising the nucleic acid chimera of claim 1 and a pharmaceutically acceptable carrier.

14. A method for immunizing a subject against West Nile Virus infection comprising administering an effective amount of the composition of claim 13 to the subject.

15. The method of claim 14, wherein the subject is selected from the group consisting of a non-human primate, a human, a horse, and a bird.

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Claim Tree

  • 1
    imera comprising a first ucleotide
    • equence encoding two structural proteins from a West Nile virus, wherein the structural proteins are premembrane/membrane (prM) and envelope (E), and a second nucleotide sequence encoding capsid (C) and nonstructural proteins from a wild-type strain of dengue virus, wherein the dengue virus is attenuated by a deletion of about 30 nucleotides from the 3′ untranslated region of the dengue genome corresponding to the TL2 stem-loop structure. 2. The nucleic acid
    • chimera of claim 1, wherein the dengue irus is
      • selected from th group consisting of dengue type 1 virus
    • chimera of claim 1, wherein the dengue irus is
      • adapted for increased growth in Vero cells. 4. The nucleic acid
    • chimera of claim 1, wherein the dengue irus is
      • dengue type 4 virus, wherein a cleavage ite is
  • 9
    omprising at least one of the
    • nucleic acid chimera of claim 1. 10. An immunogenic
  • 10
    omposition comprising the nucl ic acid ch
    • mera of claim 1 and a pharmaceutically acceptable carrier. 11. A method of ind
  • 11
    cing an immune response in a subject comprising administ ring an ef
    • ective amount of the composition of claim 10 to the subject. 12. The method of c
    • aim 11, wherein the subject is sele
      • ted from th group consisting of a non-human primate
  • 13
    ition comprising the nucl ic acid ch
    • mera of claim 1 and a pharmaceutically acceptable carrier. 14. A method for im
  • 14
    unizing a subject against West Nile Virus infection comprising administ ring an ef
    • ective amount of the composition of claim 13 to the subject. 15. The method of c
    • aim 14, wherein the subject is sele
      • ted from th group consisting of a non-human primate
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Description

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 16, 2014, is named 84407DIV_47992_ST25.txt and is 106,496 bytes in size.

FIELD OF THE INVENTION

The present invention relates to attenuated, immunogenic West Nile virus chimeras built on a dengue virus backbone for the production of immunogenic, live, attenuated West Nile virus vaccines.

BACKGROUND OF THE INVENTION

Beginning with FIG. 1A, the flavivirus genome is a single-stranded, positive-sense RNA approximately 11 kb in length, containing a 5′ untranslated region (5′ UTR); a coding region encoding the three viral structural proteins; seven nonstructural proteins, designated NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5; and a 3′ untranslated region (3′ UTR). The viral structural proteins include the capsid (C), premembrane/membrane (prM) and envelope (E) proteins. The structural and nonstructural proteins are translated as a single polyprotein. The polyprotein is then processed by cellular and viral proteases.

West Nile virus (WN) belongs to the family Flaviviridae that comprises more than 60 viruses, many of which are important human pathogens. WN is a member of the Japanese encephalitis virus (JE) serocomplex of mosquito-borne flaviviruses that includes St. Louis encephalitis, JE, and Murray Valley encephalitis viruses (Calisher, C. H. et al. 1989 J Gen Virol 70:27-43; Burke, D. S. & Monath, T. P. 2001 in: Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp, 1043-1125). Like other members of the JE antigenic complex, WN is maintained in a natural cycle that involves mosquito vectors and birds, while humans and equines are usually incidental hosts. For many years WN has been recognized as one of the most widely distributed flaviviruses with a geographic range including Africa, Australia, Europe, the Middle East and West Asia (Burke, D. S. & Monath, T. P. 2001 in: Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp. 1043-1125; Hayes, C. G. 1989 in: The Arboviruses: Epidemiology and Ecology, ed. Monath T. P. Boca Raton, Fla. CRC Press, Volume V, pp. 59-88). During 1999 WN first established itself in the USA in the Northeast and Mid-Atlantic States and more recently this virus extended its range to include the Southeastern and Western States (Anderson, J. F. et al. 1999 Science 286:2331-2333; Lanciotti, R. S. et al. 1999 Science 286:2333-2337; Campbell, G. L. et al. 2002 Lancet 2:519-529). In endemic regions, most human WN infections are asymptomatic or cause mild illness with symptoms of low-grade fever, headache, body aches, rash, myalgia, and polyarthropathy. However, human epidemics with severe disease have been reported in Israel, France, Romania, and Russia. In acute severe illness, the virus can cause hepatitis, meningitis and encephalitis leading to paralysis, and coma resulting in death. The neuropathologic lesions are similar to those of JE, with diffuse CNS inflammation and neuronal degeneration. Virus is also found in the spleen, liver, lymph nodes, and lungs of infected individuals. During the 1999 outbreak of WN in the USA, more than 60 people became ill and 7 died, while during 2002, morbidity was 3873 cases and there were 246 deaths (CDC Report: West Nile Update Current case Count, Jan. 2, 2003). Because of the recent and unexpected spread of WN from the Northeast to the Southeast and the West of the USA, this virus is considered a significant emerging disease threat that has embedded itself over a considerable region of the country. Currently, a licensed human vaccine is not available for prevention of WN disease. Mosquito control is the only practical strategy to combat the spread of disease, but effective spraying is difficult to perform in urban areas. Clearly, an effective vaccine is needed to protect at-risk populations.

Dengue viruses are mosquito-borne pathogens of the genus Flavivirus (family Flaviviridae). Four serotypes of dengue virus (DEN) have been identified, including dengue type 1 virus (DEN1), dengue type 2 virus (DEN2), dengue type 3 virus (DEN3) and dengue type 4 virus (DEN4). Live, attenuated dengue viruses of all four serotypes have been developed at Mahidol University in Thailand by passaging the wild-type viruses in primary dog kidney cell culture (Sabchareon, A. et al. 2002 Am J Trop Med Hyg 66:264-272). These are currently the least promising live, attenuated vaccine candidates for immunization against dengue virus infection and/or disease because they are not well characterized as to the relative contributions of attenuation-associated mutations to the actual mechanism of attenuation nor as to the potential for reverse mutations to revert any of the vaccine candidates to the virulent biological phenotype of the wild-type dengue virus. These vaccine candidates have been designated by a combination of their dengue serotype, the cell line through which they were passaged and the number of times they were passaged. Thus, a dengue serotype 1 wild-type virus passaged in primary dog kidney (PDK) cells 13 times is designated as DEN1 PDK13 virus. Other vaccine candidates are DEN2 PDK53, DEN3 PGMK30/FRhL3 (thirty passages in primary green monkey kidney cells, followed by three passages in fetal rhesus lung cells) and DEN4 PDK48. These four candidate vaccine viruses were derived by tissue culture passage of wild-type parental DEN1 16007, DEN2 16681, DEN3 16562 and DEN4 1036 viruses, respectively.

Except for DEN2 PDK53 virus, the number and identity of the genetic mutations that accrued during multiple passages in cell culture and that are associated with the attenuation phenotype of the vaccine candidates are unknown. Neither the relative contributions of such attenuation-associated mutations to the actual mechanism of attenuation, nor the potential for reverse mutations to revert any of the vaccine candidates to the virulent biological phenotype of the wild-type dengue virus are known for any of these four vaccine candidates. An understanding of the characteristics of a vaccine candidate is critical for the prediction of its stability and safety.

Accordingly, there is a need for attenuated, yet immunogenic flaviviruses to be used in the development of flavivirus vaccines to confer protection against flaviviruses. What would be ideal is a vaccine that would simultaneously protect an individual against flavivirus disease and be sufficiently characterized so that stability and safety are predictable.

SUMMARY THE INVENTION

Chimeric flaviviruses that are attenuated and immunogenic are provided. Chimeric viruses containing the nonstructural protein genes of a dengue virus are used as a backbone into which the structural protein genes of a West Nile virus are substituted. These chimeric viruses exhibit pronounced immunogenicity in the absence of the accompanying clinical symptoms of viral disease. The attenuated chimeric viruses are effective as immunogens or vaccines and may be combined in a pharmaceutical composition to confer immunity against West Nile virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the translation and processing of the flavivirus polyprotein. At the top is depicted the viral genome with the structural and nonstructural protein coding regions, the 5′ cap, and the 5′ and 3′ untranslated regions (UTRs) indicated. Boxes below the genome indicate precursors and mature proteins generated by the proteolytic processing cascade. Mature structural proteins are indicated by shaded boxes and the nonstructural proteins and structural protein precursors by open boxes. Contiguous stretches of uncharged amino acids are shown by black bars. Asterisks denote proteins with N-linked glycans but do not necessarily indicate the position or number of sites utilized. Cleavage sites for host signalase (♦), the viral serine protease (), furin or other Golgi-localized protease (♥), or unknown proteases (?) are indicated. Taken from Field's Virology, 2001 Fourth Edition, B. D. Lindenbach and C. M. Rice, page 998, Chapter 32.

FIG. 1B shows a strategy used to replace the genes for prM and E proteins of DEN4 with the corresponding genes of West Nile virus to produce WN/DEN4 chimeras that serve as candidate attenuated vaccine strains.

FIG. 2 shows the structure of portions of chimeric WN/DEN4 cDNAs. The top bar depicts the chimeric virus cDNA genome from the 5′ terminus of the genome to the 3′ terminus of the NS1 gene. The solid black boxes represent hydrophobic domains in the polyprotein. The vertical solid arrow indicates the position of a potential NS2B-NS3 protease cleavage site in the polyprotein between the C and prM proteins (the first junction in chimeric genome). Cleavage sites for cellular signalase are indicated by open triangles (V). A restriction enzyme-cleaved WN cDNA fragment bearing the sequence for the WN premembrane (prM) and envelope glycoprotein (E) structural protein genes was inserted into DEN4 cDNA at PstI and XhoI sites, which are underlined. The second junction is located in the COOH-terminus of the WN E protein between the two hydrophobic domains. The amino acid and nucleotide sequences of WN are presented in bold letters, and nucleotide numbering system is from GenBank accession No. AF196835. Infectivity of RNA transcripts from full-length cDNA constructs was tested by transfecting simian Vero or C6/36 mosquito cells and evaluating cell cultures for evidence of infection by immunofluorescence assay. The two clones in group 4 sustained a mutation of the amino acid+6 downstream from the cleavage site from I (isoleucine) to T (threonine) during cloning of cDNA (represented in the figure). Only these two clones were viable, yielding infectious virus following transfection of full length RNA transcripts.

*Indicates amino acids in chimeric constructs that vary at the 3+ position downstream of protease cleavage site. **Not applicable. +Two infectious chimeric WN/DEN4 viruses, namely clone 18 and 55 from group 4, were isolated.


Table of Sequences
from FIG. 2
SEQ 
SEQUENCE
ID NO
SOURCE
KKRGGRTGIA
1
WN
AAGAAAAGAGGA
2
WN
GGAAAGACCGGAATTGCA
RKRSTITLLC
3
DEN4
AGAAAAAGGTCAACG
4
DEN4
ATAACATTGCTGTGC
RKRSAVTGIA
5
WN/DEN4
AGAAAAAGGTCTGCA
6
WN/DEN4
GTGACCGGAATTGCA
RKRSAGTGIA
7
WN/DEN4
AGAAAAAGGTCTGCA
8
WN/DEN4
GGGACCGGAATTGCA
RKRSADTGIA
9
WN/DEN4
AGAAAAAGGTCTGCAG
10
WN/DEN4
ACACCGGAATTGCA
RKRSADTGTA
11
WN/DEN4
AGAAAAAGGTCTGCA
12
WN/DEN4
GACACCGGAACTGCA
INARD
13
WN
ATCAATGCTCGTGAT
14
WN
LNSRN
15
DEN4
CTGAACTCGAGGAAC
16
DEN4
INSRN
17
WN/DEN4
ATCAACTCGAGGAAC
18
WN/DEN4

FIG. 3 shows the viremia of rhesus monkeys inoculated with parental WN or DEN4 virus or their WN/DEN4 chimera or its 3′ deletion mutant WN/DEN4-3′Δ30. Twenty rhesus monkeys (Maccaca mulatta) in groups of 4 were inoculated subcutaneously (SC) with WN, DEN4, WN/DEN4 clone 18 or WN/DEN4-3′Δ30 clone 1. The quantity of virus in monkey serum was determined by direct titration in Vero cells using immunostaining focus-forming assay. Viremia was tested daily for 12 days post-inoculation for each monkey individually. Mean virus titer in serum of each monkey group shown; n is number of monkeys in group. The limit of detection of virus was 100.7 FFU/ml, and the WN/DEN4 and WN/DEN4-3′Δ30 viruses were at or below the level of detection of virus in serum.

FIG. 4. A. The Δ30 mutation removes 30 contiguous nucleotides (shaded) from the 3′ UTR of DEN4. Nucleotides are numbered from the 3′ terminus. B. Nucleotide sequence alignment of the TL2 region of DEN4 and DEN1 and their Δ30 derivatives. Also shown is the corresponding region for each of the four DEN serotypes, with upper case letters indicating sequence homology among all 4 serotypes, underlining indicating nucleotide pairing to form the stem structure. C. Predicted secondary structure of the TL2 region of each DEN serotype. Nucleotides that are removed by the Δ30 mutation for the already constructed DEN1Δ30, DEN4Δ30, DEN2Δ30 viruses are indicated (boxed) on the left and the proposed DEN3Δ30 virus is on the right (DEN1—nts 10562-10591, DEN2 Tonga/74—nts 10541-10570, DEN3 Sleman/78—nts 10535-10565, and DEN4—nts 10478-10507).


Table of Sequences from FIG. 4
SEQ
SEQUENCE
ID NO
SOURCE
GGCCCGAAGCCAGGAGGAAGCUGUAC
19
DEN4
UCCUGGUGGAAGGACUAGAGGUUAG
GGGGCCCGAAGCCAGGAGGAAGCU
20
DEN4
GUACUCCUGGUGGAAGGACUAGA
GGGGCCCAAGACUAGA
21
DEN4Δ30
GGGGCCCAACACCAGGGGAAGCU
22
DEN1
GUACCCUGGUGGUAAGGACUAGA
GGGGCCCAAGACUAGA
23
DEN1Δ30
GGGGCCCAAGGUGAGAUGAAGCU
24
DEN2
GUAGUCUCACUGGAAGGACUAGA
GGGGCCCGAGCUCUGAGGGAAGCU
25
DEN3
GUACCUCCUUGCAAAGGACUAGA
GCAGCAGCGGGGCCCAACACCAGG
26
DEN1
GGAAGCUGUACCCUGGUGGUAAGG
ACUAGAGGUUAGAGGAGACCCCCC
GCAACAACAA
AGCAAAAGGGGGCCCGAAGCCAGGA
27
DEN4
GGAAGCUGUACUCCUGGUGGAAGGA
CUAGAGGUUAGAGGAGACCCCCCCA
ACACAAAA
AGCAACAAUGGGGGCCCAAGGUGAGA
28
DEN2
UGAAGCUGUAGUCUCACUGGAAGGAC
UAGAGGUUAGAGGAGACCCCCCCAAA
ACAAAA
GCAGCAGCGGGGCCCGAGCUCUGAGG
29
DEN3
GAAGCUGUACCUCCUUGCAAAGGAC
UAGAGGUUAGAGGAGACCC 
CCCGCAAAUAAAA


Brief Description of the Sequences
GenBank Accession No. or description
DEN1
U88535
DEN2
Tonga/74 (SEQ ID No: 30 and 31)*
DEN3
Sleman/78 (SEQ ID No: 32 and 33)**
DEN4
AF326825
*DEN2 (Tonga/74) cDNA plasmid p2
Bases 1 to 10713: DEN2 virus genome cDNA:
Bases 97 to 10269: DEN2 polyprotein ORF
Bases 97 to 438: C protein ORF
Bases 439 to 936: prM protein ORF
Bases 937 to 2421: E protein ORF
Bases 2422 to 3477: NS1 protein ORF
Bases 3478 to 4131: NS2A protein ORF
Bases 4132 to 4521: NS2B protein ORF
Bases 4522 to 6375: NS3 protein ORF
Bases 6376 to 6756: NS4A protein ORF
Bases 6757 to 6825: 2K protein ORF
Bases 6826 to 7569: NS4B protein ORF
Bases 7570 to 10269: NS5 protein ORF
**DEN3 (Sleman/78) cDNA plasmid p3
Bases 1 to 10707: DEN3 virus genome cDNA
Bases 95 to 10264: DEN3 polyprotein ORF
Bases 95 to 436: C protein ORF
Bases 437 to 934: prM protein ORF
Bases 935 to 2413: E protein ORF
Bases 2414 to 3469: NS1 protein ORF
Bases 3470 to 4123: NS2A protein ORF
Bases 4124 to 4513: NS2B protein ORF
Bases 4514 to 6370: NS3 protein ORF
Bases 6371 to 6751: NS4A protein ORF
Bases 6752 to 6820: 2K protein ORF
Bases 6821 to 7564: NS4B protein ORF
Bases 7575 to 10264: NS5 protein ORF

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Immunogenic WN/DEN flavivirus chimeras and methods for preparing the WN/DEN flavivirus chimeras are provided herein. The immunogenic WN/DEN flavivirus chimeras are useful, alone or in combination, in a pharmaceutically acceptable carrier as immunogenic compositions to immunize and protect individuals and animals against infection by West Nile virus.

Chimeras of the present invention comprise nucleotide sequences encoding the immunogenic structural proteins of a West Nile virus and further nucleotide sequences selected from the backbone of a dengue virus. Chimeric viruses derived from the nucleotide sequences can be used to induce an immunogenic response against West Nile virus.

In another embodiment, the preferred chimera is a nucleic acid chimera comprising a first nucleotide sequence encoding at least one structural protein from a West Nile virus, and a second nucleotide sequence encoding nonstructural proteins from a dengue virus. In another embodiment the dengue virus is attenuated. In another embodiment the dengue virus is DEN4. In another embodiment, the structural protein can be the C protein of a West Nile virus, the prM protein of a West Nile virus, the E protein of a West Nile virus, or any combination thereof.

The term “residue” is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

Furthermore, one of skill in the art will recognize that individual substitutions, deletions or additions in the amino acid sequence, or in the nucleotide sequence encoding for the amino acids, which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified variations, wherein the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

    • 1) Alanine (A), Serine (S), Threonine (T);
    • 2) Aspartic acid (D), Glutamic acid (E);
    • 3) Asparagine (N), Glutamine (Q);
    • 4) Arginine (R), Lysine (K);
    • 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
    • 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W),

As used herein, the terms “virus chimera,”“chimeric virus,”“flavivirus chimera” and “chimeric flavivirus” means an infectious construct of the invention comprising nucleotide sequences encoding the immunogenicity of a West Nile virus and further nucleotide sequences derived from the backbone of a dengue virus.

As used herein, “infectious construct” indicates a virus, a viral construct, a viral chimera, a nucleic acid derived from a virus or any portion thereof, which may be used to infect a cell.

As used herein, “nucleic acid chimera” means a construct of the invention comprising nucleic acid comprising nucleotide sequences encoding the immunogenicity of a West Nile virus and further nucleotide sequences derived from the backbone of a dengue virus. Correspondingly, any chimeric flavivirus or flavivirus chimera of the invention is to be recognized as an example of a nucleic acid chimera.

The structural and nonstructural proteins of the invention are to be understood to include any protein comprising or any gene encoding the sequence of the complete protein, an epitope of the protein, or any fragment comprising, for example, three or more amino acid residues thereof.

Flavivirus Chimeras

West Nile virus and dengue virus are mosquito-borne flavivirus pathogens. The flavivirus genome contains a 5′ untranslated region (5′ UTR), followed by a capsid protein (C) encoding region, followed by a premembrane/membrane protein (prM) encoding region, followed by an envelope protein (E) encoding region, followed by the region encoding the nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and finally a 3′ untranslated region (3′ UTR). The viral structural proteins are C, prM and E, and the nonstructural proteins are NS1-NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

The flavivirus chimeras of the invention are constructs formed by fusing structural protein genes from a West Nile virus with non-structural protein genes from a dengue virus, e.g., DEN1, DEN2, DEN3, or DEN4.

The attenuated, immunogenic flavivirus chimeras provided herein contain one or more of the structural protein genes, or antigenic portions thereof, of the West Nile virus against which immunogenicity is to be conferred, and the nonstructural protein genes of a dengue virus.

The chimera of the invention contains a dengue virus genome as the backbone, in which the structural protein gene(s) encoding C, prM, or E protein(s) of the dengue genome, or combinations thereof, are replaced with the corresponding structural protein gene(s) from a West Nile virus that is to be protected against. The resulting chimeric virus has the properties, by virtue of being chimerized with the dengue virus, of attenuation and is therefore reduced in virulence, but expresses antigenic epitopes of the WN structural gene products and is therefore immunogenic.

The genome of any dengue virus can be used as the backbone in the attenuated chimeras described herein. The backbone can contain mutations that contribute to the attenuation phenotype of the dengue virus or that facilitate replication in the cell substrate used for manufacture, e.g., Vero cells. The mutations can be in the nucleotide sequence encoding nonstructural proteins, the 5′ untranslated region or the 3′ untranslated region, The backbone can also contain further mutations to maintain the stability of the attenuation phenotype and to reduce the possibility that the attenuated virus or chimera might revert back to the virulent wild-type virus. For example, a first mutation in the 3′ untranslated region and a second mutation in the 5′ untranslated region will provide additional attenuation phenotype stability, if desired. In particular, a mutation that is a deletion of 30 nts from the 3′ untranslated region of the DEN4 genome between nts 10478-10507 results in attenuation of the DEN4 virus (Men et al. 1996 J Virol 70:3930-3933; Durbin et al. 2001 Am J Trop Med 65:405-413). Therefore, the genome of any dengue type 4 virus containing such a mutation at this locus can be used as the backbone in the attenuated chimeras described herein. Furthermore, other dengue virus genomes containing an analogous deletion mutation in the 3′ untranslated region of the genomes of other dengue virus serotypes may also be used as the backbone structure of this invention.

Such mutations may be achieved by site-directed mutagenesis using techniques known to those skilled in the art. It will be understood by those skilled in the art that the virulence screening assays, as described herein and as are well known in the art, can be used to distinguish between virulent and attenuated backbone structures.

Construction of Flavivirus Chimeras

The flavivirus chimeras described herein can be produced by substituting at least one of the structural protein genes of the West Nile virus against which immunity is desired into a dengue virus genome backbone, using recombinant engineering techniques well known to those skilled in the art, namely, removing a designated dengue virus gene and replacing it with the desired corresponding gene of West Nile virus. Alternatively, using the sequences provided in GenBank, the nucleic acid molecules encoding the flavivirus proteins may be synthesized using known nucleic acid synthesis techniques and inserted into an appropriate vector. Attenuated, immunogenic virus is therefore produced using recombinant engineering techniques known to those skilled in the art,

As mentioned above, the gene to be inserted into the backbone encodes a West Nile virus structural protein. Preferably the West Nile virus gene to be inserted is a gene encoding a C protein, a prM protein and/or an E protein. The sequence inserted into the dengue virus backbone can encode both the prM and E structural proteins. The sequence inserted into the dengue virus backbone can encode the C, prM and E structural proteins. The dengue virus backbone is the DEN1, DEN2, DEN3, or DEN4 virus genome, or an attenuated dengue virus genome of any of these serotypes, and includes the substituted gene(s) that encode the C, prM and/or E structural protein(s) of a West Nile virus or the substituted gene(s) that encode the prM and/or E structural protein(s) of a West Nile virus. In a particular embodiment of this invention, the substituted gene that encodes the structural protein of a West Nile virus directs the synthesis of a prM protein that contains Asp and Thr, respectively, at a position 3 and 6 amino acids downstream of the cleavage site that separates the capsid protein of DEN and the premembrane protein of West Nile virus.

Suitable chimeric viruses or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of West Nile virus can be evaluated for usefulness as vaccines by screening them for phenotypic markers of attenuation that indicate reduction in virulence with retention of immunogenicity. Antigenicity and immunogenicity can be evaluated using in vitro or in vivo reactivity with West Nile antibodies or immunoreactive serum using routine screening procedures known to those skilled in the art.

Flavivirus Vaccines

The preferred chimeric viruses and nucleic acid chimeras provide live, attenuated viruses useful as immunogens or vaccines. In a preferred embodiment, the chimeras exhibit high immunogenicity while at the same time not producing dangerous pathogenic or lethal effects.

The chimeric viruses or nucleic acid chimeras of this invention can comprise the structural genes of a West Nile virus in a wild-type or an attenuated dengue virus backbone. For example, the chimera may express the structural protein genes of a West Nile virus in either of a dengue virus or an attenuated dengue virus background.

The strategy described herein of using a genetic background that contains nonstructural regions of a dengue virus genome, and, by chimerization, the properties of attenuation, to express the structural protein genes of a West Nile virus has lead to the development of live, attenuated flavivirus vaccine candidates that express structural protein genes of desired immunogenicity. Thus, vaccine candidates for control of West Nile virus pathogens can be designed.

Viruses used in the chimeras described herein are typically grown using techniques known in the art. Virus plaque or focus forming unit (FFU) titrations are then performed and plaques or FFU are counted in order to assess the viability, titer and phenotypic characteristics of the virus grown in cell culture. Wild type viruses are mutagenized to derive attenuated candidate starting materials.

Chimeric infectious clones are constructed from various flavivirus strains. The cloning of virus-specific cDNA fragments can also be accomplished, if desired. The cDNA fragments containing the structural protein or nonstructural protein genes are amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from flavivirus RNA with various primers. Amplified fragments are cloned into the cleavage sites of other intermediate clones. Intermediate, chimeric flavivirus clones are then sequenced to verify the sequence of the inserted flavivirus-specific cDNA.

Full genome-length chimeric plasmids constructed by inserting the structural or nonstructural protein gene region of flaviviruses into vectors are obtainable using recombinant techniques well known to those skilled in the art.

Method of Administration

The viral chimeras described herein are individually or jointly combined with a pharmaceutically acceptable carrier or vehicle for administration as an immunogen or vaccine to humans or animals. The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable vehicle” are used herein to mean any composition or compound including, but not limited to, water or saline, a gel, salve, solvent, diluent, fluid ointment base, liposome, micelle, giant micelle, and the like, which is suitable for use in contact with living animal or human tissue without causing adverse physiological responses, and which does not interact with the other components of the composition in a deleterious manner,

The immunogenic or vaccine formulations may be conveniently presented in viral plaque forming unit (PFU) unit or focus forming unit (FFU) dosage form and prepared by using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents commonly used by one of ordinary skill in the art.

The immunogenic or vaccine composition may be administered through different routes, such as oral or parenteral, including, but not limited to, buccal and sublingual, rectal, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. The composition may be administered in different forms, including, but not limited to, solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles and liposomes. It is expected that from about 1 to about 5 doses may be required per immunization schedule. Initial doses may range from about 100 to about 100,000 PFU or FFU, with a preferred dosage range of about 500 to about 20,000 PFU or FFU, a more preferred dosage range of from about 1000 to about 12,000 PFU or FFU and a most preferred dosage range of about 1000 to about 4000 PFU or FFU. Booster injections may range in dosage from about 100 to about 20,000 PFU or FFU, with a preferred dosage range of about 500 to about 15,000, a more preferred dosage range of about 500 to about 10,000 PFU or FFU, and a most preferred dosage range of about 1000 to about 5000 PFU or FFU. For example, the volume of administration will vary depending on the route of administration. Intramuscular injections may range in volume from about 0.1 ml to 1.0 ml.

The composition may be stored at temperatures of from about −100° C. to about 4° C. The composition may also be stored in a lyophilized state at different temperatures including room temperature. The composition may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to, filtration. The composition may also be combined with bacteriostatic agents to inhibit bacterial growth.

Administration Schedule

The immunogenic or vaccine composition described herein may be administered to humans or domestic animals, such as horses or birds, especially individuals travelling to regions where West Nile virus infection is present, and also to inhabitants of those regions. The optimal time for administration of the composition is about one to three months before the initial exposure to the West Nile virus. However, the composition may also be administered after initial infection to ameliorate disease progression, or after initial infection to treat the disease.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the chimeric virus in the immunogen or vaccine composition of this invention. Such adjuvants include, but are not limited to, the following: polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, Freund's complete adjuvant (for animals), Freund's incomplete adjuvant; sorbitan monooleate, squalene, CRL-8300 adjuvant, alum, QS 21, muramyl dipeptide, CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs, trehalose, bacterial extracts, including mycobacterial extracts, detoxified endotoxins, membrane lipids, or combinations thereof.

Nucleic Acid Sequences

Nucleic acid sequences of West Nile virus and dengue virus are useful for designing nucleic acid probes and primers for the detection of West Nile virus and dengue virus chimeras in a sample or specimen with high sensitivity and specificity. Probes or primers corresponding to West Nile virus and dengue virus can be used to detect the presence of a vaccine virus. The nucleic acid and corresponding amino acid sequences are useful as laboratory tools to study the organisms and diseases and to develop therapies and treatments for the diseases.

Nucleic acid probes and primers selectively hybridize with nucleic acid molecules encoding West Nile virus and dengue virus or complementary sequences thereof. By “selective” or “selectively” is meant a sequence which does not hybridize with other nucleic acids to prevent adequate detection of the West Nile virus sequence and dengue virus sequence. Therefore, in the design of hybridizing nucleic acids, selectivity will depend upon the other components present in the sample. The hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term “selectively hybridizes” excludes the occasional randomly hybridizing nucleic acids, and thus has the same meaning as “specifically hybridizing.” The selectively hybridizing nucleic acid probes and primers of this invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99% complementarity with the segment of the sequence to which it hybridizes, preferably 85% or more.

The present invention also contemplates sequences, probes and primers that selectively hybridize to the encoding nucleic acid or the complementary, or opposite, strand of the nucleic acid. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-species hybridization capability is maintained. By “probe” or “primer” is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes or primers can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18-24 nucleotides. Isolated nucleic acids are provided herein that selectively hybridize with the species-specific nucleic acids under stringent conditions and should have at least five nucleotides complementary to the sequence of interest as described in Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

If used as primers, the composition preferably includes at least two nucleic acid molecules which hybridize to different regions of the target molecule so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of detecting the presence of West Nile virus and dengue virus, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other organisms.

The nucleic acid sequences encoding West Nile virus and dengue virus can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism to produce recombinant West Nile virus and dengue virus peptide and/or polypeptides.

The nucleic acid sequences of the invention include a diagnostic probe that serves to report the detection of a cDNA amplicon amplified from the viral genomic RNA template by using a reverse-transcription/polymerase chain reaction (RT-PCR), as well as forward and reverse amplimers that are designed to amplify the cDNA amplicon. In certain instances, one of the amplimers is designed to contain a vaccine virus-specific mutation at the 3′-terminal end of the amplimer, which effectively makes the test even more specific for the vaccine strain because extension of the primer at the target site, and consequently amplification, will occur only if the viral RNA template contains that specific mutation.

Automated PCR-based nucleic acid sequence detection systems have been recently developed. TaqMan assay (Applied Biosystems) is widely used. A more recently developed strategy for diagnostic genetic testing makes use of molecular beacons (Tyagi and Kramer 1996 Nature Biotechnology 14:303-308). Molecular beacon assays employ quencher and reporter dyes that differ from those used in the TaqMan assay. These and other detection systems may used by one skilled in the art.

West Nile Virus/Dengue Type 4 Virus Chimeras that are Reduced in Neurovirulence and Peripheral Virulence without Loss of Immunogenicity or Protective Efficacy

A candidate live attenuated vaccine strain was constructed for West Nile virus (WN), a neurotropic flavivirus that has recently emerged in the U.S. Considerable attenuation for mice was achieved by chimerization with dengue virus type 4 (DEN4). The genes for the structural premembrane (prM) and envelope (E) proteins of DEN4 present in a full-length infectious cDNA clone were replaced by the corresponding genes of WN strain NY99. Two of 18 full-length cDNA clones of a WN/DEN4 chimera yielded full-length RNA transcripts that were infectious when transfected into susceptible cells. The two infectious clones shared a motif in the transmembrane signal domain located immediately downstream of the NS2B-NS3 protease cleavage site that separates the DEN4 capsid protein and the WN premembrane protein of the chimera. This motif, Asp and Thr at a position 3 and 6 amino acids downstream of the cleavage site, respectively, was not present in the 16 non-infectious cDNA clones. The WN/DEN4 chimera was highly attenuated in mice compared to its WN parent; the chimera was at least 28,500 times less neurovirulent in suckling mice inoculated intracerebrally and at least 10,000 times less virulent in adult mice inoculated intraperitoneally. Nonetheless, the WN/DEN4 chimera and a deletion mutant derived from it, were immunogenic and provided complete protection against lethal WN challenge. These observations provide the basis for pursuing the development of a live attenuated WN vaccine.

Recent advances in recombinant DNA technology have allowed us to develop a novel approach for constructing live attenuated flavivirus vaccines (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS USA 95:1746-1751; Pletnev, A. G. et al. 2000 Virology 274:26-31; Pletnev, A. G. et al. 2001 J Virol 75:8259-8267). Our approach was made possible by the conservation among flaviviruses of genome organization, number of viral proteins, replicative strategy, gene expression, virion structure and morphogenesis (Lindenbach, R D. & Rice, C. M. 2001 in: Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp. 1043-1125). All flaviviruses have a positive sense non-segmented RNA genome that encodes a single long polyprotein that is processed to yield capsid (C), premembrane (prM) and envelope glycoprotein (E) structural proteins followed by nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 in that order. These shared properties suggested that viable chimeric viruses could be produced by replacing the genes for the viral structural proteins in a full-length infectious cDNA clone of a flavivirus with the corresponding viral genes (in cDNA form) of another flavivirus. When tested, this strategy was successful for chimeras that contained the sequence for viral structural proteins prM and E of tick-borne encephalitis virus (TBEV) or tick-borne Langat virus (LGT), while all other sequences were derived from the full-length infectious cDNA of mosquito-borne dengue type 4 virus (DEN4). This indicated that viral structural proteins of a disparate flavivirus, TBEV or LGT, could function in the context of cis-acting 5′ and 3′ sequences and nonstructural proteins of DEN4. Significantly, both chimeras proved to be highly attenuated in mice with respect to peripheral virulence, namely, the ability of a virus to spread to the CNS from a peripheral site of inoculation and cause encephalitis. Nonetheless, the chimeras proved to be immunogenic and able to induce resistance in mice against challenge with TBEV or LGT (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS USA 95:1746-1751; Pletnev, A. G. et al. 2000 Virology 274:26-31). It appeared that a favorable balance between reduction in virus replication in vivo (attenuation) and induction of protective immunity had been achieved. We interpret this to mean that tick-borne flavivirus prM and E can interact in the context of DEN4 nonstructural proteins and cis-acting 5′ and 3′ sequences at a level sufficient for infectivity and induction of immunity but not sufficient for full expression of virulence that requires a high level of replication in vivo and ability to spread into the CNS.

Although a logical extension of this strategy was to construct WN/DEN4 chimeras, we realized that viability could not be predicted in advance because some flavivirus combinations such as some Langat virus(prM and E)/dengue virus chimeras, as well as dengue virus(prM and E)/Langat dengue virus chimeras, have not proven to be viable. Nevertheless, we were surprisingly successful in constructing viable WN/DEN4 chimeras in which the structural prM and E protein genes of the distantly related mosquito-borne WN were substituted for the corresponding genes of DEN4. We also generated a WN/DEN4 chimera with a 30 nucleotide deletion in the 3′ untranslated region (3′ UTR) that had previously been shown to render DEN4 safe but still immunogenic in adult volunteers (Durbin, A. P. et al. 2001 Am J Trop Med Hyg 65:405-413). Studies in mice were first performed to evaluate neurovirulence, peripheral virulence, immunogenicity, and protective efficacy of the newly constructed WN/DEN4 chimeric viruses.

Materials and Methods.

Cells and Viruses

Simian Vero cells (WHO seed passage 143) and mosquito C6/36 cells were obtained from Dr. L. Potash (Novavax Inc., Rockville, Md.). These Vero cells are qualified for use in production of candidate human vaccines. Simian LLCMK2 cells were purchased from the American Type Culture Collection (Manassas, Va.). Starting with West Nile virus, the WN wild-type strain NY99-35262 used in this study was kindly provided by Dr. R. Lanciotti (Centers for Disease Control and Prevention, Fort Collins, Colo.). It was originally isolated from a Chilean flamingo at the Bronx Zoo (New York) in 1999 (Lanciotti, R. S. et al. 1999 Science 286:2333-2337). The sequence of WN NY99 genome is available as GenBank accession number AF196835, per Table 1, and other strains of WN may substitute for the sequence of WN NY99 genome. A virus suspension prepared in Vero cells had a titer of 2.6×107 focus-forming units per milliliter (FFU/ml) as determined with Vero cells using an immunostaining focus-forming assay (Pletnev, A. G. 2001 Virology 282:288-300) and WN-specific mouse antibodies. Turning to dengue virus, wild-type DEN4 Caribbean strain 814669 (GenBank accession number AF326573) was used, which replicated in Vero cells with a titer of 1.1×108 FFU/ml. The sequence of recombinant DEN4 genome is available as GenBank accession number AF326825, per Table 1, and other strains of DEN4 may substitute for the sequence of DEN4 genome. The sequence of DEN1 genome is available as GenBank accession number U88536, the sequence of DEN2 genome is available as GenBank accession number M19197, and the sequence of DEN3 genome is available as GenBank accession number M93130, and any of these sequences may substitute for the sequence of DEN4 genome.

Chimeric WN/DEN4 cDNA and Recovery of Infectious Virus.

Plasmid p2A(Xhol) (Bray, M. & Lai, C.-J. 1991 PNAS USA 88:10342-10346) containing the DEN4 full-length infectious cDNA, previously employed for recovery of chimeric TBEV/DEN4 and LGT/DEN4 viruses (Pletnev, A. G. et at 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS. USA 95:1746-1751), was used for construction of WN/DEN4 cDNA. This was achieved by substituting cDNA of the WN prM and E protein genes for those of the corresponding DEN4 genes (FIG. 1B). The source of WN cDNA was a PCR product that included nucleotides (nts) 233 to 2758 of the WN strain NY99 genome. This was also kindly provided by Dr. R. Lanciotti (CDC). The nucleotide sequence of the structural protein genes in this PCR fragment was determined and compared with the published sequence of WN NY99 (GenBank accession number AF196835). Three nucleotide differences (C1893→U, C2370→U and C2385→A) were identified in the E protein sequence, none of which resulted in an amino acid substitution.

Prior experience with construction and analysis of tick-borne/DEN4 chimeras indicated that we could not predict a priori the sequence of the DEN4 C protein/tick-borne flavivirus prM protein junction required for viability (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS. USA 95:1746-1751). For this reason, we adopted an empirical approach and tested several different C/prM junction sequences (FIG. 2). This was not necessary for the downstream junction because it was located within the COOH-terminal region of WN E. Initially, 3 sets of C/prM junctions were tested but only one yielded a viable WN/DEN4 chimera (FIG. 2). The primers employed for construction of the chimeras by PCR used oligonucleotide 5′-TCAAAACAAAAGAAAAGATCTGCAGTGACCGGAATTGCAGTCATGATTGGC-3′ (SEQ ID NO: 34), or 5′-TCAAAACAAAAGAAAAGATCTGCAGGGACCGGAATTGCAGTCATGATTGGC-3′ (SEQ ID NO: 35), or 5′-TCAAAACAAAAGAAAAGATCTGCAGACACCGGAATTGCAGTCATGATTGGC-3′ (SEQ ID NO: 36) as a forward primer and oligonucleotide 5′-CCGCAAGAAACGTCATAGCAATTGACCTGTCACTCGAGTTGATTCCCATCCACAA CAGAAGAGC-3′ (SEQ ID NO: 37) as a reverse primer. Stable full-length WN/DEN4 cDNA clones were identified after transformation of E. coli BD 1528 with a ligation mixture that contained the PCR product and the vector both of which were digested by PstI and XhoI (FIG. 2). Sequences at the junctions between WN and DEN4 genes in each chimeric plasmid were verified.

Plasmid DNA containing full-length WN/DEN4 cDNA was linearized with Asp718. In vitro RNA synthesis and transfection of cells with its RNA transcripts were performed as described previously (Pletnev, A. G. 2001 Virology 282:288-300). Briefly, RNA transcripts of full-length WN/DEN4 constructs listed in FIG. 2 were used to transfect simian LLCMK2, simian Vero cells or mosquito C6/36 cells in the presence of LipofectAmine 2000 reagent (GIBCO BRL, Gaithersburg, Md.) in a BSL-3 laboratory generously provided by Dr. L. Markoff (CBER, FDA). Transfected cells were examined by immunofluorescence assay (IFA) for the presence of WN or DEN4 proteins using a WN- or DEN4-specific hyperimmune mouse ascitic fluid (HMAF). Two infectious chimeric viruses containing WN/DEN4 group 4 junctions (FIG. 2), namely, WN/DEN4 clone 18 and 55, were isolated. The recovered chimeras were amplified once in simian Vero or mosquito C6/36 cells, viral RNA was isolated and then reverse transcribed into cDNA that was used for sequence analysis (Table 1). In a similar manner, the sequence of the Vero cell-derived WN/DEN4 clone 18 was determined after an additional purification by two rounds of terminal end-point dilution and amplification in Vero cells infected at a multiplicity of 0.01. The resulting virus suspension had a titer of 1.7×106 FFU/ml.

To introduce a deletion into the 3′ untranslated region (UTR) of WN/DEN4 genome, the DNA fragment between the XhoI site (nt 2345 of DEN4 genome; GenBank accession number AF326827) and the Asp718 site at the 3′ end of plasmid WN/DEN4-18 DNA was replaced by the corresponding Xhol-Asp718-fragment derived from full-length cDNA of a DEN4 mutant, clone p4Δ30 (Durbin et al. 2001 Am, J Trop Med. Hyg 65:405-413). This mutant had 30 nts deleted from the 3′ untranslated region (UTR) of the genome between nts 10478-10507. Full-length RNA generated by SP6 polymerase from 10 different plasmids was tested for infectivity by transfection of simian Vero cells. Two individual WN/DEN4-3′Δ30 cDNA clones were infectious. The rescued deletion mutants, WN/DEN4-3′Δ30 clone 1 and 78, were purified twice by terminal end-point dilution and amplified in Vero cells to a titer of 1.4×105 and 6×104 FFU/ml, respectively. Virol RNA was isolated, and complete sequence of the 3′ deletion mutant genome was determined (Table 1),

Evaluation of Parental and Chimeric Viruses in Mice

Neurovirulence of Vero cell culture-propagated parental WN (strain NY99), parental DEN4 (strain 814669), chimeric WN/DEN4 (clone 18) and its deletion mutant (clone 1) was evaluated in a BSL-3 facility. Three-day-old Swiss Webster mice (Taconic Farms) in groups of 9 to 12 were inoculated by the intracerebral (IC) route with decimal dilutions ranging from 0.1 to 105 FFU of virus in 0.03 ml of MEM/0.25% human serum albumin. Mice were observed for 21 days for development of fatal encephalitis. The 50% lethal dose (LD50) of each virus was determined by the method of Reed and Muench (Reed, L. J. & Muench, H. 1938 Am. J Hyg 27:493-497). Parental and chimeric viruses were also analyzed for peripheral virulence by intraperitoneal (IP) inoculation of 3-week-old Swiss female mice in groups of 10. Mice were inoculated with decimal dilutions of virus ranging from 0.1 to 105 FFU and observed for 28 days for fatal encephalitis. Moribund mice were humanely euthanized.

Mice that survived IP inoculation were bled on day 28 to evaluate the WN-specific neutralizing antibody response. Serum from mice in each group was pooled and the WN virus-neutralizing antibody titer of the serum pool was determined by FFU reduction assay in Vero cells as described previously (Pletnev, A. G. et al. 2001 J Virol 75:8259-8267; Pletnev, A. G. 2001 Virology 282:288-300). Briefly, a 1:10 dilution of pooled sera was prepared in MEM containing 2% fetal bovine serum (FBS) and then heat inactivated for 30 min at 56° C. Serial twofold dilutions of inactivated pooled sera were mixed with an equal volume of a virus suspension containing approximately 50 FFU of WN. The mixture was incubated for 30 min at 37° C., and 0.4 ml was then added to duplicate wells of Vero cells in a 6-well plate. After 1 h of absorption at 37° C., the inoculum was removed and cells were overlaid with MEM containing 2% FBS, 50 μg/ml gentamycin, 0.25 fungizone, and 1% tragacanth gum. Antibody titer was determined after 2 days of incubation by an immunostaining focus-forming assay (Pletnev, A. G. 2001 Virology 282:288-300) that used WN-specific HMAF. Neutralizing antibody titer was the highest dilution of pooled sera that reduced focus formation 50% compared to sera collected from non-immunized mice.

The surviving mice were challenged IP on day 29 with 100 IP LD50 (103 FFU) of parental WN virus and observed for fatal encephalitis for a period of 21 days. Moribund mice were humanely euthanized.

Results.

Construction and Recovery of Chimeric WN/DEN4 Viruses

In total we constructed 18 plasmids that contained full-length chimeric WN/DEN4 cDNA which included the structural prM and E protein genes of the WN strain NY99 with all other sequences derived from DEN4 (FIG. 2). Full-length RNA generated by SP6 RNA polymerase from only 2 of the 18 chimeric cDNAs was infectious when transfected into mosquito C6/36 or simian Vero cells. Evidence for virus infectivity was detected by IFA. In the case of the 2 viable chimeric viruses, 80-100% of transfected cells were infected by day 5 as indicated by IFA using WN-specific HMAF. The 2 viable chimeric viruses (WN/DEN4 clones 18 and 55) had the C/prM intergenic junction sequence of group 4 chimera shown in FIG. 2, i.e., +3 Asp and +6 Thr amino acids downstream of the cleavage site, respectively. The presence of this junction was confirmed by sequence analysis of the recovered chimeras. Also, the complete genomic sequence of the two chimeras rescued from cDNA in Vero cells was determined and compared with the consensus sequence of their parental WN NY99 and DEN4 viruses as well as the nucleotide sequence of the WN/DEN4 viral chimera insert in the plasmid DNA from which infectious RNA transcripts were derived (Table 1). Analysis of plasmid DNAs revealed 4 differences in nucleotide sequence from the consensus WN sequence determined by RT-PCR of a high titered suspension of WN strain NY99. Three of these differences produced amino acid substitutions in prM (Ile6→Thr and Ile146→Val) and E (Thr282→Ala). In addition, variability between (i) Glu92 and Asp and (ii) Leu112 and Ser was identified in the DEN4 NS3 and NS4B nonstructural proteins of the WN/DEN4 clone 55. Also, sequence of the Vero cell-grown WN/DEN4 clone 18 differed from its progenitor plasmid cDNA sequence in the DEN4 NS4B gene. A change U7162→C that caused the substitution Leu112→Ser was identified, which was observed previously (Blaney, J. E. et al. 2001 J Virol 75:9731-9740). Interestingly, a different substitution at this locus, Leu112→Phe, was also previously observed by Blaney et al. upon passage of wild-type DEN4 in Vero cells.

Following our success in constructing full-length infectious WN/DEN4 cDNAs, we constructed chimeric virus mutants with a 30 nucleotide deletion in their 3′ untranslated region (UTR). Two mutants, WN/DEN4-3′Δ30 clone 1 and clone 78, were recovered from transfected Vero cells. The complete sequence of both these clones was analyzed (Table 1). Sequence of clone 78 differed from the sequence of plasmid DNA from which its infectious RNA transcripts were derived. A change of C7141→U produced an amino acid substitution Thr105→Ile in the NS4B protein. The WN/DEN4-3′Δ30 clone 1 also exhibited only one nucleotide difference from the plasmid cDNA sequence. This resulted in the same NS4B amino acid change (Leu112→Ser) that was observed in WN/DEN4 clone 18.

The WN/DEN4 chimera replicated more efficiently in Vero cells than did WN/DEN4-3′Δ30. The unmodified WN/DEN4 chimera reached a titer of 106 FFU/ml on day 6 in cells infected with a multiplicity of infection of 0.01; this was approximately 10-fold higher than the titer attained by the deletion mutant by day 6. The titer of the unmodified chimera was nearly the same as that attained by parental DEN4 under the same conditions.

Mouse Neurovirulence.

Before evaluating chimeric viruses for virulence in mice, the Vero cell-rescued chimeric WN/DEN4 virus and its 3′ deletion mutant were cloned biologically twice by terminal end-point dilution and then amplified in qualified Vero cells. The titer attained by the Vero cell-adapted WN/DEN4 clone 18 and WN/DEN4-3′Δ30 clone 1 was 1.7×106 FFU/ml and 1.4×105FFU/ml, respectively.

Both chimeric WN/DEN4 virus and the deletion mutant WN/DEN4-3′Δ30 as well as parental WN strain NY99 and DEN4 strain 814669 viruses were evaluated in 3-day-old Swiss mice for neurovirulence by direct IC inoculation (Table 2). Wild-type WN NY99 grown in Vera cells was highly neurovirulent with an intracerebral LD50 of 0.35 FFU in suckling Swiss mice. Wild-type DEN4 also grown in Vero cells was less neurovirulent with an IC LD50 of 407 FFU. Both WN/DEN4 and WN/DEN4-3′Δ30 chimeric viruses exhibited a significant reduction in neurovirulence compared to their WN and DEN4 parents. All of the mice inoculated IC with 103 FFU of WN/DEN4 or its 3° deletion mutant survived during a 21 day observation period. At a higher dose of 104 FFU, only 4 of 11 mice inoculated with WN/DEN4 died. Thus, in suckling mice the WN/DEN4 chimera was more than 28,571 times less neurovirulent than its WN parent. The chimera with the 30 nt deletion was also significantly less neurovirulent than its WN parent. These observations are consistent with earlier observations that chimerization of TBEV or LGT with DEN4 significantly reduced their neurovirulence for mice (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev. A. G. & Men, R. 1998 PNAS. USA 95:1746-1751; Pletnev, A. G., Bray, M. & Lai, C.-J. 1993 J Virol 67:4956-4963).

Peripheral Virulence in Mice

Subsequently, we evaluated the chimeric viruses for peripheral virulence, i.e., the ability of virus inoculated by the IP route to spread from a peripheral site to the central nervous system and cause encephalitis. Both chimeras were highly attenuated compared to their WN parent (Table 2 and 3). Notably, IP inoculation of 104 FFU of the deletion mutant chimera or 105 FFU of the unmodified chimera did not induce fatal encephalitis in any of the 3-week-old Swiss mice, whereas the IP LD50 for the WN parent was 10 FFU.

The chimeras were also evaluated in adult SCID mice because previous studies of tick-borne flaviviruses and their DEN4 chimeras indicated that SCID mice were a more sensitive detector of peripheral virulence than immunocompetent mice. Intraperitoneal inoculation of the maximum quantity of chimera, 105 FFU for WN/DEN4 and 104 FFU for WN/DEN4-3′Δ30, did not produce encephalitis in any instance (Table 2). In contrast, the IP LD50 for parental WN was 6 FFU. These observations confirmed that the ablation of peripheral virulence of the WN chimeras had been achieved.

Immunogenicity and Protective Efficacy of Chimeric Viruses in Mice

The two chimeras were immunogenic; a single IP inoculation of 102 FFU of the WN/DEN4 chimera induced a moderate level of serum WN neutralizing antibodies (1:93), while a 10-fold higher concentration (103 FFU) induced a very high titer of WN neutralizing antibodies (1:1189) (Table 3). Also, 103 FFU of the chimeric WN/DEN4-3′Δ30 deletion mutant stimulated a high level of such antibodies (1:292). Intraperitoneal challenge of the immunized mice on day 29 with 100 IP LD50 (103 FFU) of parental WN indicated that the chimeras provided 90 to 100% protection against this high dose WN challenge (Table 3). There was a good correlation between the titer of serum WN neutralizing antibodies that developed in response to immunization and the degree of resistance induced. All unvaccinated control mice developed signs of CNS disease 7 to 13 days after challenge with 100 IP LD50 of WN and these animals died shortly thereafter. To determine whether there was an age-related resistance of mice to WN, another group of 7-week-old mice also served as controls; they were the same age as immunized mice at time of challenge. This group of older control mice was challenged with one IP LD50, determined in 3-week-old mice. Seven of eight mice died during the 21 day observation period. This indicated that age-dependent resistance of mice to WN was not a factor in the observed protective effect of immunization.


TABLE 1
Mutations that were identified in genome of the WN/DEN4 or WN/DEN4-3′Δ30
chimera during cloning and rescue of chimera from cDNA in simian Vero cells
Region
WN/DEN4
WN/DEN4-3′Δ30
of
NT
Recombinant virus
Recombinant virus
Virus
genome
(position)*
pDNA**
clone 55
clone 18
pDNA+
clone 1
clone 78
Amino acid change
WN
prM
U428
C
C
C
C
C
C
Ile6 → Thr
A847
G
G
G
G
G
G
Ile146 → Val
E
A1566
G
G
G
G
G
G
silent
A1810
G
G
G
G
G
G
Thr282→ Ala
DEN4
NS3
A4799
A
C/a
A
A
A
A
Glu92 → Asp
NS4B
C7141
C
C
C
C
C
U
Thr105 → Ile
U7162
U
C/u
C
U
C
U
Leu112→ Ser
*Numbering of nucleotide sequence of structural protein genes derived from the sequence of WN NY99 genome (GenBank accession number AF196835) and numbering of nucleotide sequence of nonstructural protein genes derived from the sequence of DEN4 genome (GenBank accession number AF326825).
**Plasmid DNA.
+Comparison of the pDNA for the parental cDNA clones used to derive the chimeric viruses are indicated in Durbin, A. et al. 2001 Am J Trop Med Hyg 65: 405-413


TABLE 2
Neurovirulence and peripheral virulence of parental West Nile virus (WN) or Dengue type
4 virus (DEN4) and their chimeric WN/DEN4 virus or its 3′ deletion mutant WN/DEN4-
3′Δ30 in mice as assayed by intracerebral (IC) or intraperitoneal (IP) inoculation
Neurovirulence:
Peripheral virulence:
LD50 (FFU) after
Reduction
LD50 (FFU) after
LD50 (FFU) after
Reduction
IC inoculation of 3-
from WN
IP inoculation of 3-
IP inoculation of 3-
from WN
Virus
day-old Swiss mice
parent
week-old Swiss mice
week-old SCID mice
parent
DEN4
  407
>100,000*
>100,000*
WN
    0.35
   10
    6.0
WN/DEN4
>10,000*
>28,571x
>100,000*
>100,000*
>10,000x
Chimera
(clone 18)
WN/DEN4-
 >1,000*
 >2,857x
 >10,000*
 >10,000*
 >1,000x
3′Δ30
Chimera
(clone 1)
Note:
Each decimal dilution was tested in 9 to 12 mice in group.
*Highest concentration tested.


TABLE 3
Peripheral virulence, antibody response and protective efficacy
of parental (WN or DEN4) viruses and chimeric WN/DEN4 virus or
its 3′ deletion mutant WN/DEN4-3′Δ30 in 3-week-old Swiss mice
Mean titer
Mortality after
of WN
survivors
neutralizing
inoculated IP
Mice
Dose
Mortality
antibody in
with 100 IP
inoculated
(FFU*)
after IP
pooled sera
LD50 of WN
IP with
inoculated
inoculation
on day 28
on day 29
WN
0.1
0/10
<1:10 
10/10 (100%)
1
0/10
1:24
10/10 (100%)
10
5/10
1:40
 4/5 (80%)
100
10/10 
1,000
9/10
10,000
10/10 
WN/DEN4
1
0/10
1:26
10/10 (100%)
Chimera
10
0/10
1:21
9/10 (90%)
(clone 18)
100
0/10
1:93
7/10 (70%)
1,000
0/10
 1:1189
0/10 (0%) 
10,000
0/10
 1:585
0/9** (0%) 
100,000
0/10
 1:924
0/10 (0%) 
WN/DEN4-
1
0/10
1:28
9/10 (90%)
3′Δ30
10
0/10
<1:10 
9/10 (90%)
Chimera
100
0/10
1:14
8/10 (80%)
(clone 1)
1,000
0/10
 1:292
1/10 (10%)
10,000
0/10
 1:269
0/10 (0%) 
DEN4
1,000
0/10
<1:10 
10/10 (100%)
10,000
0/10
1:13
8/10 (80%)
100,000
0/10
1:22
10/10 (100%)
Control
<1:10 
10/10 (100%)
*Focus forming unit.
**One of the 10 mice inoculated died as a result of trauma; WN virus was not detected in the brain by tissue culture (Vero cell) assay.


TABLE 4
Chimeric WN/DEN4 and its 3′ deletion mutant WN/DEN4-
3′Δ30 are attenuated in rhesus monkeys
Viremia
Mean peak titer
Virus
of viremia
inoculated
Dose of
No. of
Mean
during 2 weeks
subcuta-
virus
monkeys
No.
duration
post-inoculation
neously
(FFU)
inoculated
viremic
(days)
log10 (FFU/ml)*
WN/DEN4
105
4
3
1.5
0.78
106
4
2
0.5
<0.7
WN/DEN4-
105
4
0
0
<0.7
3′Δ30
WN
105
2
2
5.5
2.63
106
2
2
5.5
2.76
DEN4
106
4
4
3.8
2.23
*Tested daily for 10 days.
Note:
0.7 log10(FFU/ml) is a lowest level of detectable viremia in serum. 0.6 log10(FFU/ml) was used to calculate mean peak titer of viremia for animals that had no detectable viremia.


TABLE 5
Immunogenicity and protective efficacy of chimeric WN/DEN4 and its 3′
deletion mutant WN/DEN4-3′Δ30 in rhesus monkeys
No. of monkeys
Geo. mean titer
viremic during
Group of monkeys
of WN serum
2 weeks post
inoculated
neutralizing
challenge with
SC with
No. of
antibody on post
105 FFU of WN
Dose
mon-
immunization day
(Mean peak titer;
Virus
(FFU)
keys
42 (range)
log10 FFU/ml)*
WN/
105
4
1:661
(416-1126)
0
DEN4
106
4
1:501
(270-727)
0
WN/
105
4
1:186
(109-247)
0
DEN4-
3′Δ30
WN
105
2
1:1318
(1305-1324)
0
106
2
1:708
(599-842)
0
DEN4
106
4
<1:20
4 (2.04**)
*Tested daily for 10 days.
**Mean duration of viremia was 3.75 days.

Attenuation, Immunogenicity and Protective Efficacy of West Nile/DEN4 Chimeras in Rhesus Monkeys.

It has been established that some non-human primates are readily infected with a number of flaviviruses by the peripheral route (Simmons et al. 1931 Philipp J Sci 44:1-247; Rosen, 1958 Am J Trop Med Hyg 7:406-410). Thus, infection of monkeys represents the closest experimental system to flavivirus infection of humans. The response of monkeys to flavivirus infection is similar to that of humans in that there is a four to six day viremia, although lower primates do not usually develop clinical flavivirus symptoms. The objectives of flavivirus studies in monkeys are: (1) to evaluate the immunogenicity of various candidate vaccines; (2) to evaluate the infectivity and virulence (attenuation phenotype) of candidate vaccines as measured by the duration of viremia in days and the peak virus titer in FFU/ml; and (3) to evaluate the protective efficacy of the candidate vaccines against challenge by wild-type flavivirus.

1) Inoculation: Each monkey is inoculated with a total of 105 or 106 FFU of virus diluted in L15 medium with SPG (Durbin, A. P. et al. 2001 Am J Trap Med Hyg 65:405-413). Normally, virus is inoculated by the subcutaneous route to anesthetized animals.

2) Blood collection: Following inoculation of virus, blood samples of 3.0 ml are taken daily for two weeks and 5.0 ml at 3 weeks, 4 weeks, 5 weeks, and 6 weeks.

3) Challenge with parental wild-type flavivirus: Where virus challenge is deemed appropriate to evaluate the protective efficacy, monkeys are inoculated with wild-type virus at 105 FFU/dose in a 1.0 ml volume subcutaneously in the upper arm area,

4) Laboratory assays: Serum samples are collected to be used to determine: (a) the duration and level of viremia by direct viral plaque or FFU assay; and (b) the titer of neutralizing antibodies induced as measured by FFU reduction neutralization test, all tests well known to those skilled in the art of vaccine development.

Attenuation, immunogenicity, and protective efficacy of the West Nile/DEN4 chimeras were studied in 20 rhesus monkeys (Tables 4 and 5). Eight monkeys were inoculated subcutaneously (SC) with WN/DEN4 (clone 18); 4 animals received 105 FFU, while the other 4 received 106 FFU. Four monkeys were inoculated SC with 105 FFU of WN/DEN4-3′Δ30 (clone 1). A group of 4 monkeys was inoculated SC with parental West Nile virus; 2 animals received 105 FFU, while the other received 106 FFU. Finally, another group of 4 monkeys was inoculated SC with 106 of DEN4 (Table 4).

Each of the monkeys inoculated SC with 105 or 106 FFU of West Nile virus developed a viremia that lasted 5 to 6 days and attained a mean peak titer of 2.6 to 2.8 log10 (FFU/ml) (FIG. 3, Table 4). In contrast, WN/DEN4 induced viremia in only 5 of the 8 monkeys inoculated with 105 or 106 FFU. Viremia lasted only one to two days and attained a peak titer 100 fold lower than observed for WN infected monkeys. Significantly, each of the 4 monkeys inoculated SC with 105 FFU of the WN/DEN4-3′Δ30 mutant failed to develop a detectable viremia.

Although the WN/DEN chimera and its deletion mutant were significantly attenuated for rhesus monkeys, these hybrid viruses induced a moderate to high level of serum WN neutralizing antibodies in each immunized animal (Table 5). The two chimeras also induced complete resistance to SC challenge with 105 FFU of West Nile virus on day 42 post immunization. Viremia of WN was not detected in any of the 12 monkeys immunized with WN/DEN4 or its deletion mutant. The West Nile challenge virus replicated efficiently in monkeys previously infected with DEN4 virus. This indicates that the high level of protection against WN challenge afforded by infection with WN/DEN4 chimeric viruses is specified by the WN protective antigens in the chimera and not by the DEN4 component of the chimera.

The Δ30 mutation was first described and characterized in the DEN4 virus (Men, R. et al. 1996J Virol 70:3930-7). In DEN4, the mutation consists of the removal of 30 contiguous nucleotides comprising nucleotides 10478-10507 of the 3′ UTR (FIG. 4A) which form a putative stem-loop structure referred to as TL2 (Proutski, V. et al. 1997 Nucleic Acids Res 25:1194-202). Among the flaviviruses, large portions of the UTR form highly conserved secondary structures (Hahn, C. S., et al. 1987J Mol Biol 198:33-41; Proutski, V. et al. 1997 Nucleic Acids Res 25:1194-202). Although the individual nucleotides are not necessarily conserved in these regions, appropriate base pairing preserves the stem-loop structure in each serotype, a fact that is not readily apparent when only considering the primary sequence (FIG. 4B, C). We have demonstrated that the Δ30 mutation specifies an attenuation phenotype that is transportable to other DEN serotypes, DEN1 (Whitehead, S. S. et al. 2003 J Virol 77:1653-1657) and DEN2 (Tonga/74) (U.S. Provisional application, filed Dec. 23, 2002, as NIH-1230.002PR). This indicates that the Δ30 mutation is expected to have a corresponding effect on DEN3 wild-type virus. We envision constructing this remaining virus by deletion of the TL2 region of the virus, e.g., DEN3 (Sleman/78) (FIG. 4C). These attenuated or wild type DEN1, DEN2, or DEN3 viruses could readily replace the DEN4 wild type or DEN4-3′Δ30 viruses presented in these examples.

These findings specifically identify two candidate WN live attenuated virus vaccines. The first, WN/DEN4, is about 100-fold attenuated in comparison to WN wild-type virus as indicated by the greatly restricted level of viremia. The second virus, WN/DEN4-3′Δ30, is more attenuated than WN/DEN4 as indicated by the absence of viremia in monkey serum and by the moderately decreased serum neutralizing antibody response. Thus, the methods and viruses taught provide live attenuated WN vaccines of differing levels of attenuation, each of which is highly protective against wild-type WN virus challenge. Similar attenuated WN/DEN chimeric viruses on a DEN1, DEN2, or DEN3 background are envisioned,

Further Attenuation of WN/DEN4 Chimeras by Introduction of Additional Mutations in the Genes for the Non-Structural Proteins of DEN4 that Serve as a Component of these Vaccine Candidates.

We contemplate achieving an increase in the level of attenuation of the candidate vaccine WN/DEN4 or WN/DEN4-3′Δ30 chimera if need be by adding one or more attenuation mutations to the DEN4 component of the chimeras. A large set of mutations that attenuate DEN4 in mice (Blaney, et al. 2001 J Virol 75:9731-9740; Blaney, et al. 2002 Virology 300:125-139; Hanley, et al. 2002 J Virol 76:525-31) has been identified in the part of the DEN4 genome included in the WN/DEN4 chimeric viruses. Members from this set of attenuating mutations can be introduced in the WN/DEN4 chimeric virus to further attenuate these viruses. For example, it might be necessary to further attenuate the WN/DEN4 virus, which possesses some residual neurovirulence as indicated above. The feasibility of this approach to achieve further attenuation is exemplified by introducing a viable mutation that specifies a temperature sensitive phenotype as well as a phenotype of growth restriction in suckling mouse brain into the non-structural protein 3 (NS3) of the DEN4 component of the WN/DEN4 chimera. Mutation 4891 (isoleucine→threonine) had previously been identified at nucleotide 4891 of the NS3 gene of DEN4 (Blaney, et al. 2002 Virology 300:125-139). Mutation 4891 specified two desirable phenotypes, i.e., temperature sensitivity and growth restriction in brain tissue. Similarly, mutation 4995 (serine→proline), also in NS3, specified the same two desirable phenotypes (Blaney, et al. 2001 J Virology 75:9731-9740, 2001). The 4891 and 4995 mutations also increase replication fitness of DEN4 in Vero cells, i.e., they are Vero cell adaptation mutations. The wild type amino acid residue at DEN4 4891 (isoleucine) is conserved in DEN2 Tonga/74 and DEN3 Sleman/78, but not DEN1 West Pacific. The wild type amino acid residue at DEN4 4995 (serine) is conserved in DEN1 West Pacific, DEN2 Tonga/74, but not DEN3 Sleman. One or both of these mutations may also be included in a WN/DEN1, 2, or 3 chimera. Thus, their inclusion in WN/DEN4 virus is contemplated as achieving an increase in replication of the virus in Vero cells or the genetic stability of the mutation during manufacture in Vero cells.

DISCUSSION

initially, we demonstrated that although prM and E proteins of distantly related tick-borne and mosquito-borne flaviviruses are highly divergent, these proteins could be interchanged in some instances without loss of virus viability (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS USA 95:1746-1751). This approach has been used to create new chimeric flaviviruses (Bray, M., Men, R. & Lai, C.-J. 1996 J. Virol. 70:4162-4166; Chambers, T. J. et al. 1999 J Virol 73:3095-3101; Guirakhoo, F. et al. 2000 J Virol 74:5477-5485; Huang, C. Y. et al. 2000 J Virol 74:3020-3028; Van Der Most, R. G. et al. 2000 J Virol 74:8094-8101; Caufour, P. S. et al. 2001 Virus Res 79:1-14).

Previously, we succeeded in constructing and recovering viable tick-borne/DEN4 chimeras (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS USA 95:1746-1751; Pletnev, A. G., Bray, M. & Lai, C.-J. 1993 J Virol 67:4956-4963). In these instances, the tick-borne flavivirus parent was tick-borne encephalitis virus, a highly virulent virus, or Langat virus, a naturally attenuated tick-borne virus. Thus, the two components of these chimeras had disparate vector hosts, namely ticks and in the case of DEN4, mosquitoes. Decreased efficiency of gene product interactions in the chimeras was thought to be the basis for the marked attenuation exhibited by these hybrid viruses. Nonetheless, although highly attenuated in mice, the TBEV/DEN4 and LGT/DEN4 chimeras were immunogenic and provided considerable protection against their parental tick-borne flavivirus. In the present instance, both virus parents of the WN/DEN4 chimeras are transmitted by mosquitoes. However, vector preference differs, Aedes for DEN4 and Culex for WN (Burke, D. S. & Monath, T. P. 2001 in Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp. 1043-1125; Hayes, C. G. 1989 in The Arboviruses: Epidemiology and Ecology, ed. Monath T. P. Boca Raton, F. L.: CRC Press, Volume V, pp. 59-88).

In the present study, we constructed viable WN/DEN4 chimeras that contained a DEN4 genome whose genes for structural prM and E proteins were replaced by the corresponding genes of WN strain NY99. Among flaviviruses, the hydrophobic domain between C and prM (“transmembrane signal domain”) varies in sequence and also varies in length from 14 to 20 amino acids (Stocks, C. E. & Lobigs, M. 1998 J Virol 72:2141-2149). It acts as a signal sequence for translocation of prM protein into the endoplasmic reticulum lumen where post-translation maturation of this protein occurs (Lindenbach, B. D. & Rice, C. M. 2001 in Fields Virology, eds. Knipe, D. M. & Howley, P. M. Lippincott Williams and Wilkins, Philadelphia, 4-th ed., pp. 1043-1125). This signal peptide is flanked at its NH2-terminal region by the viral protease NS2B-NS3 cleavage site and at its COOH-terminal region by a cellular signalase cleavage site. Four different junctions at the protease cleavage site between DEN4 C and WN prM protein were introduced separately in chimeric constructs (FIG. 2). The C/prM fusion sequence at the viral protease cleavage site (KRS) in the chimeras was constructed to be similar to that of the DEN4 parent, which provides its NS2B-NS3 protease for the processing of the chimeric polyprotein. However, each of the chimeric constructs of group 1 and 2 chimeras contain a unique substitution in the transmembrane signal sequence at the third amino acid position downstream of the protease cleavage site, while another sequence is shared by group 3 and group 4 chimeras (FIG. 1A, FIG. 2). Thus, the transmembrane signal of the constructs is similar in length but exhibits polymorphism for group 1, group 2 and groups 3 and 4 together. This occurs at the third amino acid position downstream of the protease cleavage site. Viable WN/DEN4 virus was recovered only when construct number 4 (FIG. 2) was employed to prepare RNA transcripts for transfection. Infectious virus was recovered from 2 of 5 separate clones that encoded Asp in the 3+ amino acid position. And only the 2 clones that also contained a second-site mutation at the 6+ amino acid position downstream of the protease cleavage site that substituted Thr for Ile were infectious; this mutation occurred during cloning of cDNA in bacteria (FIG. 2, Table 1). In contrast, none of the 13 clones that encoded Gly or Val at the 3+ amino acid position produced infectious virus following transfection. This suggests that the transmembrane signal sequence between C and prM is a determinant of viability in the context of a WN/DEN4 chimera. This is consistent with an earlier observation made with yellow fever virus that the transmembrane signal sequence between C and prM protein plays a role in viability and neurovirulence (Lee, E. et al. 20001 Virol, 74:24-32),

The +3 and +6 Asp and Thr motif at the capsid protein—preM protein cleavage site that was required for viability of the chimera could not be predicted from the sequence of either parent, i.e., DEN4 and West Nile virus, because neither parent had this +3 and +6 motif. Success was achieved by testing a number of disparate sequences at the cleavage site and this led to the identification of the +3 and +6 Asp and Thr motif that was required for viability. For this reason, we advocate an empirical approach that embraces testing several different C/prM junction sequences for identification of other motifs that produce equally viable chimeric virus.

The WN strain NY99 exhibited considerable virulence in Swiss mice; its IC LD50 was 0.35 FFU for suckling mice and its IP LD50 was 10 FFU for 3-week-old Swiss mice (Table 2). Nearly the same level of neurovirulence was observed for a wild-type strain of WN isolated in Israel that was evaluated in CD-1 (ICR) mice: IC LD50 and IP LD50 were estimated to be 1.1 and 4.3 PFU, respectively (Halevy, M. et al. 1994 Arch Virol 137:355-370). In addition, a high degree of genomic similarity (>99.8%) between the WN NY99 and the WN Israel-1998 was recently confirmed by sequence analysis (Lanciotti, R. S. et al. 1999 Science 286:2333-2337) indicating that both highly pathogenic strains of WN, representing North American and Middle Eastern viruses, are closely related. Wild-type DEN4 Caribbean strain 814669 was moderately neurovirulent for suckling mice with an IC LD50 of 407 FFU, and it was approximately 20 times more virulent than its cDNA cloned virus (Pletnev, A. G. & Men, R. 1998 PNAS USA 95:1746-1751). In contrast, the WN/DEN4 chimera and its 3′ deletion mutant were significantly less neurovirulent than their wild-type DEN4 or WN parent. Only at a high dose of 104 FFU did a minority of mice, inoculated IC with WN/DEN4 chimera, die. Also, the WN/DEN4 chimera inoculated IC at this dose caused death of suckling mice later than parental WN virus: 4-5 days post-infection for wild-type WN compared to 9-13 days post-infection for the chimera. Additional methods and procedures are taught that allow further attenuation of the IC virulence of the WN/DEN4 chimeric virus by the introduction of mutations that are known to attenuate DEN4 virus for the brain of mice. In addition, we also contemplate achieving further attenuation of WN/DEN4-3′Δ30 by the incorporation of additional attenuating mutations.

Despite the high peripheral virulence of wild-type WN strain NY99 (IP LD50 of 10 FFU), chimerization of WN with DEN4 completely ablated this property of its WN parent. Thus, 3-week-old Swiss mice survived IP inoculation of 104 or 105 FFU of chimeric virus. Our observations are consistent with earlier findings that a similar large reduction of peripheral neurovirulence of TBEV or LGT occurs following chimerization with DEN4 (Pletnev, A. G. et al. 1992 PNAS USA 89:10532-10536; Pletnev, A. G. & Men, R. 1998 PNAS. USA 95:1746-1751; Pletnev, A. G., Bray, M. & Lai, C.-J. 1993 J Virol 67:4956-4963). Similar observations were made when the WN/DEN4 chimeras were tested in SCID mice for peripheral virulence (Table 2).

Although highly attenuated, the WN/DEN4 chimeras stimulated a moderate to high level of serum neutralizing antibodies against WN NY99 (Table 3). There was a strong correlation between the level of neutralizing antibodies to WN induced by immunization and resistance to subsequent lethal WN challenge. The immune response of mice inoculated with the chimeras was dose-dependent and indicated that the unmodified WN/DEN4 chimera was slightly more immunogenic than the corresponding 3′ deletion mutant. However, 90 to 100% protection against WN challenge was achieved when a single 103 FFU dose of WN/DEN4 chimera or its 3′ deletion mutant was used for immunization. A higher dose (104 FFU) of either chimera provided complete protection to WN challenge. The WN/DEN4 and WN/DEN4-3′Δ30 were also highly attenuated, immunogenic, and protective against WN virus challenge in non-human primates (rhesus monkeys). Thus, the WN prM and E proteins of the chimeric viruses represent effective antigens able to induce complete protection to challenge with highly virulent WN in both mice and monkeys. Our observations concerning safety, immunogenicity, and protective efficacy of the chimeric WN/DEN4 vaccine candidates in mice and monkeys provide a basis for extending our evaluation of the vaccine candidates to humans and to domestic animals, such as horses or birds, which are at high risk. In this way, the use of the WN/DEN4 chimeras as vaccines is envisioned for humans and domestic animals, such as horses or birds.

* * *

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.

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<213> ORGANISM: Dengue 1

<400> SEQENCE: 22

ggggcccaac accaggggaa gcuguacccu ggugguaagg acuaga 46

<210> SEQ ID NO: 23

<211> LENGTH: 16

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Dengue1 delta 30

<400> SEQENCE: 23

ggggcccaag acuaga 16

<210> SEQ ID NO: 24

<211> LENGTH: 46

<212> TYPE: DNA

<213> ORGANISM: Dengue 2

<400> SEQENCE: 24

ggggcccaag gugagaugaa gcuguagucu cacuggaagg acuaga 46

<210> SEQ ID NO: 25

<211> LENGTH: 47

<212> TYPE: DNA

<213> ORGANISM: Dengue 3

<400> SEQENCE: 25

ggggcccgag cucugaggga agcuguaccu ccuugcaaag gacuaga 47

<210> SEQ ID NO: 26

<211> LENGTH: 82

<212> TYPE: DNA

<213> ORGANISM: Dengue 1

<400> SEQENCE: 26

gcagcagcgg ggcccaacac caggggaagc uguacccugg ugguaaggac uagagguuag 60

aggagacccc ccgcaacaac aa 82

<210> SEQ ID NO: 27

<211> LENGTH: 83

<212> TYPE: DNA

<213> ORGANISM: Dengue 4

<400> SEQENCE: 27

agcaaaaggg ggcccgaagc caggaggaag cuguacuccu gguggaagga cuagagguua 60

gaggagaccc ccccaacaca aaa 83

<210> SEQ ID NO: 28

<211> LENGTH: 84

<212> TYPE: DNA

<213> ORGANISM: Dengue 2

<400> SEQENCE: 28

agcaacaaug ggggcccaag gugagaugaa gcuguagucu cacuggaagg acuagagguu 60

agaggagacc cccccaaaac aaaa 84

<210> SEQ ID NO: 29

<211> LENGTH: 83

<212> TYPE: DNA

<213> ORGANISM: Dengue 3

<400> SEQENCE: 29

gcagcagcgg ggcccgagcu cugagggaag cuguaccucc uugcaaagga cuagagguua 60

gaggagaccc cccgcaaaua aaa 83

<210> SEQ ID NO: 30

<211> LENGTH: 15159

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Dengue2 (Tonga/74) plasmid p2

<400> SEQENCE: 30

agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta 60

gttctaactg ttttttgatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120

agaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcaac tgtacaacag 180

ttgacaaaga gattctcact tggaatgctg cagggacgag gaccactaaa attgttcatg 240

gccctggtgg cattccttcg tttcctaaca atcccaccaa cagcagggat attaaaaaga 300

tggggaacaa ttaaaaaatc aaaggctatt aatgttctga gaggcttcag gaaagagatt 360

ggaaggatgc tgaatatctt aaacaggaga cgtagaactg taggcatgat catcatgctg 420

actccaacag tgatggcgtt tcatctgacc acacgcaacg gagaaccaca catgattgtc 480

agtagacaag aaaaagggaa aagccttctg ttcaagacaa aggatggcac gaacatgtgt 540

accctcatgg ccatggacct tggtgagttg tgtgaagaca caatcacgta taaatgtcct 600

tttctcaagc agaacgaacc agaagacata gattgttggt gcaactccac gtccacatgg 660

gtaacttatg ggacatgtac caccacagga gagcacagaa gagaaaaaag atcagtggcg 720

cttgttccac acgtgggaat gggattggag acacgaactg aaacatggat gtcatcagaa 780

ggggcctgga aacatgccca gagaattgaa acttggattc tgagacatcc aggctttacc 840

ataatggccg caatcctggc atacaccata gggacgacgc atttccaaag agtcctgata 900

ttcatcctac tgacagccat cgctccttca atgacaatgc gctgcatagg aatatcaaat 960

agggactttg tggaaggagt gtcaggaggg agttgggttg acatagtttt agaacatgga 1020

agttgtgtga cgacgatggc aaaaaacaaa ccaacactgg actttgaact gataaaaaca 1080

gaagccaaac aacctgccac cttaaggaag tactgtatag aggccaaact gaccaacacg 1140

acaacagact cgcgctgccc aacacaaggg gaacccaccc tgaatgaaga gcaggacaaa 1200

aggtttgtct gcaaacattc catggtagac agaggatggg gaaatggatg tggattgttt 1260

ggaaaaggag gcatcgtgac ctgtgctatg ttcacatgca aaaagaacat ggaaggaaaa 1320

attgtgcagc cagaaaacct ggaatacact gtcgtgataa cacctcattc aggggaagaa 1380

catgcagtgg gaaatgacac aggaaaacat ggtaaagaag tcaagataac accacagagc 1440

tccatcacag aggcggaact gacaggctat ggcactgtta cgatggagtg ctctccaaga 1500

acgggcctcg acttcaatga gatggtgttg ctgcaaatgg aagacaaagc ctggctggtg 1560

cacagacaat ggttcctaga cctaccgttg ccatggctgc ccggagcaga cacacaagga 1620

tcaaattgga tacagaaaga aacactggtc accttcaaaa atccccatgc gaaaaaacag 1680

gatgttgttg tcttaggatc ccaagagggg gccatgcata cagcactcac aggggctacg 1740

gaaatccaga tgtcatcagg aaacctgctg ttcacaggac atctcaagtg caggctgaga 1800

atggacaaat tacaacttaa agggatgtca tactccatgt gcacaggaaa gtttaaaatt 1860

gtgaaggaaa tagcagaaac acaacatgga acaatagtca ttagagtaca atatgaagga 1920

gacggctctc catgcaagat cccctttgag ataatggatc tggaaaaaag acatgttttg 1980

ggccgcctga tcacagtcaa cccaattgta acagaaaagg acagtccagt caacatagaa 2040

gcagaacctc cattcggaga cagctacatc atcataggag tggaaccagg acaattgaag 2100

ctggactggt tcaagaaagg aagttccatc ggccaaatgt ttgagacaac aatgagggga 2160

gcgaaaagaa tggccatttt gggtgacaca gcctgggatt ttggatctct gggaggagtg 2220

ttcacatcaa taggaaaggc tctccaccag gtttttggag caatctacgg ggctgctttc 2280

agtggggtct catggactat gaagatcctc ataggagtta tcatcacatg gataggaatg 2340

aactcacgta gcactagtct gagcgtgtca ctggtgttag tgggaatcgt gacactttac 2400

ttgggagtta tggtgcaggc cgatagtggt tgcgttgtga gctggaagaa caaagaacta 2460

aaatgtggca gtggaatatt cgtcacagat aacgtgcata catggacaga acaatacaag 2520

ttccaaccag aatccccttc aaaactggcc tcagccatcc agaaagcgca tgaagagggc 2580

atctgtggaa tccgctcagt aacaagactg gaaaatctta tgtggaaaca gataacatca 2640

gaattgaatc atattctatc agaaaatgaa gtgaaactga ccatcatgac aggagacatc 2700

aaaggaatca tgcaggtagg aaaacgatct ttgcggcctc aacccactga gttgaggtat 2760

tcatggaaaa catggggtaa agcgaaaatg ctctccacag aactccacaa tcagaccttc 2820

ctcattgatg gtcccgaaac agcagaatgc cccaacacaa acagagcttg gaattcactg 2880

gaagttgagg actacggctt tggagtattc actaccaata tatggctaag attgagagaa 2940

aagcaggatg tattttgtga ctcaaaactc atgtcagcgg ccataaagga caacagagcc 3000

gtccatgctg atatgggtta ttggatagaa agcgcactca atgatacatg gaagatagag 3060

aaagcttctt tcattgaagt caaaagttgc cactggccaa agtcacacac cctatggagt 3120

aatggagtgc tagaaagcga gatggtcatt ccaaagaatt tcgctggacc agtgtcacaa 3180

cataataaca gaccaggcta ttacacacaa acagcaggac cttggcatct aggcaagctt 3240

gagatggact ttgatttctg cgaagggact acagtggtgg taaccgagaa ctgtggaaac 3300

agagggccct ctttaagaac aaccactgcc tcaggaaaac tcataacgga atggtgttgt 3360

cgatcttgca cactaccacc actaagatac agaggtgagg atggatgttg gtacgggatg 3420

gaaatcagac cattgaaaga gaaagaagaa aatctggtca gttctctggt tacagccgga 3480

catgggcaga ttgacaattt ctcattagga atcttgggaa tggcactgtt ccttgaagaa 3540

atgctcagga ctcgagtagg aacaaaacat gcaatattac tcgtcgcagt ttctttcgtg 3600

acgctaatca cagggaacat gtcttttaga gacctgggaa gagtgatggt tatggtgggt 3660

gccaccatga cagatgacat aggcatgggt gtgacttatc tcgctctact agcagctttt 3720

agagtcagac caacctttgc agctggactg ctcttgagaa aactgacctc caaggaatta 3780

atgatgacta ccataggaat cgttcttctc tcccagagta gcataccaga gaccattctt 3840

gaactgaccg acgcgttagc tctaggcatg atggtcctca agatggtgag aaacatggaa 3900

aaatatcagc tggcagtgac catcatggct attttgtgcg tcccaaatgc tgtgatatta 3960

cagaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtctgtttc ccccctgctc 4020

ttaacatcct cacaacagaa agcggactgg ataccattag cgttgacgat caaaggtctt 4080

aatccaacag ccatttttct aacaaccctc tcaagaacca acaagaaaag gagctggcct 4140

ttaaatgagg ccatcatggc ggttgggatg gtgagtatct tggccagctc tctcttaaag 4200

aatgacatcc ccatgacagg accattagtg gctggagggc tccttactgt gtgctacgtg 4260

ctaactgggc ggtcagccga tctggaatta gagagagcta ccgatgtcaa atgggatgac 4320

caggcagaga tatcaggtag cagtccaatc ctgtcaataa caatatcaga agatggcagc 4380

atgtcaataa agaatgaaga ggaagagcaa acactgacta tactcattag aacaggattg 4440

cttgtgatct caggactctt tccggtatca ataccaatta cagcagcagc atggtatctg 4500

tgggaagtaa agaaacaacg ggctggagtg ctgtgggatg tcccctcacc accacccgtg 4560

ggaaaagctg aattggaaga tggagcctac agaatcaagc aaaaaggaat ccttggatat 4620

tcccagatcg gagctggagt ttacaaagaa ggaacatttc acacaatgtg gcacgtcaca 4680

cgtggcgctg tcctaatgca taaggggaag aggattgaac catcatgggc ggacgtcaag 4740

aaagacttaa tatcatatgg aggaggttgg aagctagaag gagaatggaa agaaggagaa 4800

gaagtccagg tcttggcatt ggagccaggg aaaaatccaa gagccgtcca aacaaagcct 4860

ggccttttta gaaccaacac tggaaccata ggtgccgtat ctctggactt ttcccctggg 4920

acgtcaggat ctccaatcgt cgacaaaaaa ggaaaagttg taggtctcta tggcaatggt 4980

gtcgttacaa ggagtggagc atatgtgagt gccatagctc agactgaaaa aagcattgaa 5040

gacaatccag agattgaaga tgacatcttt cgaaagagaa gattgactat catggatctc 5100

cacccaggag caggaaagac aaagagatac ctcccggcca tagtcagaga ggccataaaa 5160

agaggcttga gaacactaat cctagccccc actagagtcg tggcagctga aatggaggaa 5220

gcccttagag gacttccaat aagataccaa actccagcta tcagggctga gcacaccggg 5280

cgggagattg tagacttaat gtgtcatgcc acatttacca tgaggctgct atcaccaatc 5340

agggtgccaa attacaacct gatcatcatg gacgaagccc attttacaga tccagcaagc 5400

atagcagcta ggggatacat ctcaactcga gtggagatgg gggaggcagc tggaattttt 5460

atgacagcca ctcctccggg tagtagagat ccatttcctc agagcaatgc accaattatg 5520

gacgaagaaa gagaaattcc ggaacgttca tggaactctg ggcacgagtg ggtcacggat 5580

tttaaaggaa agactgtctg gtttgttcca agcataaaaa ccggaaatga catagcagcc 5640

tgcctgagaa agaatggaaa gagggtgata caactcagta ggaagacctt tgattctgaa 5700

tatgtcaaga ctagaaccaa tgactgggat ttcgtggtta caactgacat ctcggaaatg 5760

ggcgccaact ttaaagctga gagggtcata gaccccagac gctgcatgaa accagttata 5820

ttgacagacg gcgaagagcg ggtgattctg gcaggaccca tgccagtgac ccactctagt 5880

gcagcacaaa gaagagggag aataggaagg aatccaagga atgaaaatga tcaatatata 5940

tatatggggg aaccactgga aaatgatgaa gactgtgcgc actggaagga agctaagatg 6000

ctcctagata atatcaacac acctgaagga atcattccca gcttgttcga gccagagcgt 6060

gaaaaggtgg atgccattga cggtgaatat cgcttgagag gagaagcacg gaaaactttt 6120

gtggacctaa tgagaagagg agacctacca gtctggttgg cttataaagt ggcagctgaa 6180

ggtatcaact acgcagacag aagatggtgt tttgacggaa ccagaaacaa tcaaatcttg 6240

gaagaaaatg tggaagtgga aatctggaca aaggaagggg aaaggaaaaa attgaaacct 6300

agatggttag atgctaggat ctactccgac ccactggcgc taaaagagtt caaggaattt 6360

gcagccggaa gaaagtccct aaccctgaac ctaattacag agatgggcag actcccaact 6420

tttatgactc agaaggccag agatgcacta gacaacttgg cggtgctgca cacggctgaa 6480

gcgggtggaa aggcatacaa tcatgctctc agtgaattac cggagaccct ggagacattg 6540

cttttgctga cactgttggc cacagtcacg ggaggaatct tcctattcct gatgagcgga 6600

aggggtatgg ggaagatgac cctgggaatg tgctgcataa tcacggccag catcctctta 6660

tggtatgcac aaatacagcc acattggata gcagcctcaa taatattgga gttctttctc 6720

atagtcttgc tcattccaga accagaaaag cagaggacac ctcaggataa tcaattgact 6780

tatgtcatca tagccatcct cacagtggtg gccgcaacca tggcaaacga aatgggtttt 6840

ctggaaaaaa caaagaaaga cctcggactg ggaaacattg caactcagca acctgagagc 6900

aacattctgg acatagatct acgtcctgca tcagcatgga cgttgtatgc cgtggctaca 6960

acatttatca caccaatgtt gagacatagc attgaaaatt cctcagtaaa tgtgtcccta 7020

acagccatag ctaaccaagc cacagtgcta atgggtctcg gaaaaggatg gccattgtca 7080

aagatggaca ttggagttcc cctccttgct attgggtgtt actcacaagt caaccctata 7140

accctcacag cggctcttct tttattggta gcacattatg ccatcatagg accgggactt 7200

caagccaaag caactagaga agctcagaaa agagcagcag cgggcatcat gaaaaaccca 7260

actgtggatg gaataacagt gatagatcta gatccaatac cctatgatcc aaagtttgaa 7320

aagcagttgg gacaagtaat gctcctagtc ctctgcgtga cccaagtgct gatgatgagg 7380

actacgtggg ctttgtgtga agccttaact ctagcaactg gacccgtgtc cacattgtgg 7440

gaaggaaatc cagggagatt ctggaacaca accattgcag tgtcaatggc aaacatcttt 7500

agagggagtt acctggctgg agctggactt ctcttttcta tcatgaagaa cacaaccagc 7560

acgagaagag gaactggcaa tataggagaa acgttaggag agaaatggaa aagcagactg 7620

aacgcattgg ggaaaagtga attccagatc tacaaaaaaa gtggaattca agaagtggac 7680

agaaccttag caaaagaagg cattaaaaga ggagaaacgg atcatcacgc tgtgtcgcga 7740

ggctcagcaa aactgagatg gttcgttgaa aggaatttgg tcacaccaga agggaaagta 7800

gtggaccttg gttgtggcag agggggctgg tcatactatt gtggaggatt aaagaatgta 7860

agagaagtta aaggcttaac aaaaggagga ccaggacacg aagaacctat ccctatgtca 7920

acatatgggt ggaatctagt acgcttacag agcggagttg atgttttttt tgttccacca 7980

gagaagtgtg acacattgtt gtgtgacata ggggaatcat caccaaatcc cacggtagaa 8040

gcgggacgaa cactcagagt cctcaaccta gtggaaaatt ggctgaacaa taacacccaa 8100

ttttgcgtaa aggttcttaa cccgtacatg ccctcagtca ttgaaagaat ggaaacctta 8160

caacggaaat acggaggagc cttggtgaga aatccactct cacggaattc cacacatgag 8220

atgtactggg tgtccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaaga 8280

atgctgatca acagattcac tatgagacac aagaaggcca cctatgagcc agatgtcgac 8340

ctcggaagcg gaacccgcaa tattggaatt gaaagtgaga caccgaacct agacataatt 8400

gggaaaagaa tagaaaaaat aaaacaagag catgaaacgt catggcacta tgatcaagac 8460

cacccataca aaacatgggc ttaccatggc agctatgaaa caaaacagac tggatcagca 8520

tcatccatgg tgaacggagt agtcagattg ctgacaaaac cctgggacgt tgttccaatg 8580

gtgacacaga tggcaatgac agacacaact ccttttggac aacagcgcgt cttcaaagag 8640

aaggtggata cgagaaccca agaaccaaaa gaaggcacaa aaaaactaat gaaaatcacg 8700

gcagagtggc tctggaaaga actaggaaag aaaaagacac ctagaatgtg taccagagaa 8760

gaattcacaa aaaaggtgag aagcaatgca gccttggggg ccatattcac cgatgagaac 8820

aagtggaaat cggcgcgtga agccgttgaa gatagtaggt tttgggagct ggttgacaag 8880

gaaaggaacc tccatcttga agggaaatgt gaaacatgtg tatacaacat gatggggaaa 8940

agagagaaaa aactaggaga gtttggtaaa gcaaaaggca gcagagccat atggtacatg 9000

tggctcggag cacgcttctt agagtttgaa gccctaggat ttttgaatga agaccattgg 9060

ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcataa gctaggttac 9120

atcttaagag aggtgagcaa gaaagaagga ggagcaatgt atgccgatga caccgcaggc 9180

tgggacacaa gaatcacaat agaggatttg aaaaatgaag aaatgataac gaaccacatg 9240

gcaggagaac acaagaaact tgccgaggcc atttttaaat tgacgtacca aaacaaggtg 9300

gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360

caaaggggta gtggacaagt tggcacctat ggcctcaaca ctttcaccaa catggaagca 9420

caactaatta ggcaaatgga gggggaagga atcttcaaaa gcatccagca cttgacagcc 9480

tcagaagaaa tcgctgtgca agattggcta gtaagagtag ggcgtgaaag gttgtcaaga 9540

atggccatca gtggagatga ttgtgttgtg aaacctttag atgatagatt tgcaagagct 9600

ctaacagctc taaatgacat gggaaaggtt aggaaggaca tacagcaatg ggagccctca 9660

agaggatgga acgactggac gcaggtgccc ttctgttcac accattttca cgagttaatt 9720

atgaaagatg gtcgcacact cgtagttcca tgcagaaacc aagatgaatt gatcggcaga 9780

gcccgaattt cccagggagc tgggtggtct ttacgggaga cggcctgttt ggggaagtct 9840

tacgcccaaa tgtggagctt gatgtacttc cacagacgtg atctcaggct agcggcaaat 9900

gccatctgct cggcagtccc atcacactgg attccaacaa gccggacaac ctggtccata 9960

cacgccagcc atgaatggat gacgacggaa gacatgttga cagtttggaa cagagtgtgg 10020

atcctagaaa atccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080

tacctgggaa aaagagaaga ccaatggtgc ggctcgctga ttgggctgac aagcagagcc 10140

acctgggcga agaatatcca gacagcaata aaccaagtca gatccctcat tggcaatgag 10200

gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaggcagga 10260

gttttgtggt agaaaaacat gaaacaaaac agaagtcagg tcggattaag ccatagtacg 10320

ggaaaaacta tgctacctgt gagccccgtc caaggacgtt aaaagaagtc aggccatttt 10380

gatgccatag cttgagcaaa ctgtgcagcc tgtagctcca cctgagaagg tgtaaaaaat 10440

ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc ggttagagga 10500

gacccctccc ttacagatcg cagcaacaat gggggcccaa ggtgagatga agctgtagtc 10560

tcactggaag gactagaggt tagaggagac ccccccaaaa caaaaaacag catattgacg 10620

ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca ggacgccaga 10680

aaatggaatg gtgctgttga atcaacaggt tctggtaccg gtaggcatcg tggtgtcacg 10740

ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 10800

atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 10860

taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 10920

catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 10980

atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc 11040

acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 11100

aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 11160

ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 11220

cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 11280

atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 11340

ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 11400

ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 11460

tcgtcttcaa gaattctcat gtttgacagc ttatcatcga taagctttaa tgcggtagtt 11520

tatcacagtt aaattgctaa cgcagtcagg caccgtgtat gaaatctaac aatgcgctca 11580

tcgtcatcct cggcaccgtc accctggatg ctgtaggcat aggcttggtt atgccggtac 11640

tgccgggcct cttgcgggat atcgtccatt ccgacagcat cgccagtcac tatggcgtgc 11700

tgctggcgct atatgcgttg atgcaatttc tatgcgcacc cgttctcgga gcactgtccg 11760

accgctttgg ccgccgccca gtcctgctcg cttcgctact tggagccact atcgactacg 11820

cgatcatggc gaccacaccc gtcctgtgga tcctctacgc cggacgcatc gtggccggca 11880

tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag 11940

atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc 12000

ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg 12060

tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag 12120

agcgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg 12180

gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg 12240

tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga 12300

tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg 12360

gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccgacg 12420

cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc cccattatga 12480

ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg tccaggcagg 12540

tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc agcctaactt 12600

cgatcactgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc acatggaacg 12660

ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg ttgcgtcgcg 12720

gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg ctaacggatt 12780

caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg cgcaaaccaa 12840

cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc ggcgcatctc 12900

gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg 12960

gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac 13020

gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg 13080

tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg 13140

gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa 13200

gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg 13260

tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag 13320

catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga 13380

ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca 13440

gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg 13500

tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga 13560

tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 13620

ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 13680

cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca 13740

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 13800

aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 13860

gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 13920

gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 13980

cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 14040

aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 14100

tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 14160

ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 14220

ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 14280

cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 14340

ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 14400

gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 14460

atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 14520

aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 14580

aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 14640

gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 14700

cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 14760

gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 14820

tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 14880

ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 14940

ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 15000

atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 15060

cgcaacgttg ttgccattgc tgcaagatct ggctagcgat gaccctgctg attggttcgc 15120

tgaccatttc cgggcgcgcc gatttaggtg acactatag 15159

<210> SEQ ID NO: 31

<211> LENGTH: 3391

<212> TYPE: PRT

<213> ORGANISM: Dengue 2 (Tonga/74)

<400> SEQENCE: 31

Met Asn Asn Gln Arg Lys Lys Ala Arg Asn Thr Pro Phe Asn Met Leu

1 5 10 15

Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg

20 25 30

Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met

35 40 45

Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly

50 55 60

Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val

65 70 75 80

Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn

85 90 95

Arg Arg Arg Arg Thr Val Gly Met Ile Ile Met Leu Thr Pro Thr Val

100 105 110

Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val

115 120 125

Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Lys Asp Gly

130 135 140

Thr Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu

145 150 155 160

Asp Thr Ile Thr Tyr Lys Cys Pro Phe Leu Lys Gln Asn Glu Pro Glu

165 170 175

Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly

180 185 190

Thr Cys Thr Thr Thr Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala

195 200 205

Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp

210 215 220

Met Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Ile Glu Thr Trp

225 230 235 240

Ile Leu Arg His Pro Gly Phe Thr Ile Met Ala Ala Ile Leu Ala Tyr

245 250 255

Thr Ile Gly Thr Thr His Phe Gln Arg Val Leu Ile Phe Ile Leu Leu

260 265 270

Thr Ala Ile Ala Pro Ser Met Thr Met Arg Cys Ile Gly Ile Ser Asn

275 280 285

Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val

290 295 300

Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr

305 310 315 320

Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu

325 330 335

Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Asp Ser

340 345 350

Arg Cys Pro Thr Gln Gly Glu Pro Thr Leu Asn Glu Glu Gln Asp Lys

355 360 365

Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly

370 375 380

Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Thr

385 390 395 400

Cys Lys Lys Asn Met Glu Gly Lys Ile Val Gln Pro Glu Asn Leu Glu

405 410 415

Tyr Thr Val Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly

420 425 430

Asn Asp Thr Gly Lys His Gly Lys Glu Val Lys Ile Thr Pro Gln Ser

435 440 445

Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu

450 455 460

Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln

465 470 475 480

Met Glu Asp Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu

485 490 495

Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile

500 505 510

Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln

515 520 525

Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu

530 535 540

Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr

545 550 555 560

Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly

565 570 575

Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Ile Val Lys Glu Ile

580 585 590

Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly

595 600 605

Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys

610 615 620

Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu

625 630 635 640

Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser

645 650 655

Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asp Trp Phe

660 665 670

Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly

675 680 685

Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser

690 695 700

Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe

705 710 715 720

Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys

725 730 735

Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser

740 745 750

Thr Ser Leu Ser Val Ser Leu Val Leu Val Gly Ile Val Thr Leu Tyr

755 760 765

Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys

770 775 780

Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Thr Asp Asn Val

785 790 795 800

His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys

805 810 815

Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Gly Ile Cys Gly Ile

820 825 830

Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Ser

835 840 845

Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met

850 855 860

Thr Gly Asp Ile Lys Gly Ile Met Gln Val Gly Lys Arg Ser Leu Arg

865 870 875 880

Pro Gln Pro Thr Glu Leu Arg Tyr Ser Trp Lys Thr Trp Gly Lys Ala

885 890 895

Lys Met Leu Ser Thr Glu Leu His Asn Gln Thr Phe Leu Ile Asp Gly

900 905 910

Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu

915 920 925

Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu

930 935 940

Arg Leu Arg Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser

945 950 955 960

Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp

965 970 975

Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe

980 985 990

Ile Glu Val Lys Ser Cys His Trp Pro Lys Ser His Thr Leu Trp Ser

995 1000 1005

Asn Gly Val Leu Glu Ser Glu Met Val Ile Pro Lys Asn Phe Ala

1010 1015 1020

Gly Pro Val Ser Gln His Asn Asn Arg Pro Gly Tyr Tyr Thr Gln

1025 1030 1035

Thr Ala Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp

1040 1045 1050

Phe Cys Glu Gly Thr Thr Val Val Val Thr Glu Asn Cys Gly Asn

1055 1060 1065

Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile

1070 1075 1080

Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr

1085 1090 1095

Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu

1100 1105 1110

Lys Glu Lys Glu Glu Asn Leu Val Ser Ser Leu Val Thr Ala Gly

1115 1120 1125

His Gly Gln Ile Asp Asn Phe Ser Leu Gly Ile Leu Gly Met Ala

1130 1135 1140

Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His

1145 1150 1155

Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly

1160 1165 1170

Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly

1175 1180 1185

Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala

1190 1195 1200

Leu Leu Ala Ala Phe Arg Val Arg Pro Thr Phe Ala Ala Gly Leu

1205 1210 1215

Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile

1220 1225 1230

Gly Ile Val Leu Leu Ser Gln Ser Ser Ile Pro Glu Thr Ile Leu

1235 1240 1245

Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met

1250 1255 1260

Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala

1265 1270 1275

Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys

1280 1285 1290

Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Leu

1295 1300 1305

Leu Thr Ser Ser Gln Gln Lys Ala Asp Trp Ile Pro Leu Ala Leu

1310 1315 1320

Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu

1325 1330 1335

Ser Arg Thr Asn Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile

1340 1345 1350

Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys

1355 1360 1365

Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu

1370 1375 1380

Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu

1385 1390 1395

Glu Arg Ala Thr Asp Val Lys Trp Asp Asp Gln Ala Glu Ile Ser

1400 1405 1410

Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser

1415 1420 1425

Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu

1430 1435 1440

Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser

1445 1450 1455

Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys

1460 1465 1470

Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Val

1475 1480 1485

Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys

1490 1495 1500

Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu

1505 1510 1515

Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu

1520 1525 1530

Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys

1535 1540 1545

Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu

1550 1555 1560

Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly

1565 1570 1575

Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Arg Thr

1580 1585 1590

Asn Thr Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly

1595 1600 1605

Thr Ser Gly Ser Pro Ile Val Asp Lys Lys Gly Lys Val Val Gly

1610 1615 1620

Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser

1625 1630 1635

Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile

1640 1645 1650

Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu

1655 1660 1665

His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val

1670 1675 1680

Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro

1685 1690 1695

Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu

1700 1705 1710

Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Glu His Thr Gly

1715 1720 1725

Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg

1730 1735 1740

Leu Leu Ser Pro Ile Arg Val Pro Asn Tyr Asn Leu Ile Ile Met

1745 1750 1755

Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly

1760 1765 1770

Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe

1775 1780 1785

Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser

1790 1795 1800

Asn Ala Pro Ile Met Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser

1805 1810 1815

Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr

1820 1825 1830

Val Trp Phe Val Pro Ser Ile Lys Thr Gly Asn Asp Ile Ala Ala

1835 1840 1845

Cys Leu Arg Lys Asn Gly Lys Arg Val Ile Gln Leu Ser Arg Lys

1850 1855 1860

Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp

1865 1870 1875

Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys

1880 1885 1890

Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile

1895 1900 1905

Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro

1910 1915 1920

Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg

1925 1930 1935

Asn Pro Arg Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro

1940 1945 1950

Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met

1955 1960 1965

Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Leu

1970 1975 1980

Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr

1985 1990 1995

Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg

2000 2005 2010

Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Lys Val Ala Ala Glu

2015 2020 2025

Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Thr Arg

2030 2035 2040

Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr

2045 2050 2055

Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala

2060 2065 2070

Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe

2075 2080 2085

Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met

2090 2095 2100

Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu

2105 2110 2115

Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Lys Ala

2120 2125 2130

Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu

2135 2140 2145

Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu

2150 2155 2160

Phe Leu Met Ser Gly Arg Gly Met Gly Lys Met Thr Leu Gly Met

2165 2170 2175

Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile

2180 2185 2190

Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu

2195 2200 2205

Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln

2210 2215 2220

Asp Asn Gln Leu Thr Tyr Val Ile Ile Ala Ile Leu Thr Val Val

2225 2230 2235

Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys

2240 2245 2250

Lys Asp Leu Gly Leu Gly Asn Ile Ala Thr Gln Gln Pro Glu Ser

2255 2260 2265

Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu

2270 2275 2280

Tyr Ala Val Ala Thr Thr Phe Ile Thr Pro Met Leu Arg His Ser

2285 2290 2295

Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn

2300 2305 2310

Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser

2315 2320 2325

Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser

2330 2335 2340

Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Leu Leu Leu Val

2345 2350 2355

Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr

2360 2365 2370

Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro

2375 2380 2385

Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr

2390 2395 2400

Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val

2405 2410 2415

Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu

2420 2425 2430

Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Val Ser Thr Leu Trp

2435 2440 2445

Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser

2450 2455 2460

Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu

2465 2470 2475

Leu Phe Ser Ile Met Lys Asn Thr Thr Ser Thr Arg Arg Gly Thr

2480 2485 2490

Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu

2495 2500 2505

Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly

2510 2515 2520

Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg

2525 2530 2535

Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu

2540 2545 2550

Arg Trp Phe Val Glu Arg Asn Leu Val Thr Pro Glu Gly Lys Val

2555 2560 2565

Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly

2570 2575 2580

Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly

2585 2590 2595

Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn

2600 2605 2610

Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Val Pro Pro

2615 2620 2625

Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro

2630 2635 2640

Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu

2645 2650 2655

Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Val Lys Val

2660 2665 2670

Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Arg Met Glu Thr Leu

2675 2680 2685

Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg

2690 2695 2700

Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn

2705 2710 2715

Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg

2720 2725 2730

Phe Thr Met Arg His Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp

2735 2740 2745

Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Thr Pro

2750 2755 2760

Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu

2765 2770 2775

His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr

2780 2785 2790

Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala

2795 2800 2805

Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp

2810 2815 2820

Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr

2825 2830 2835

Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg

2840 2845 2850

Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr

2855 2860 2865

Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg

2870 2875 2880

Met Cys Thr Arg Glu Glu Phe Thr Lys Lys Val Arg Ser Asn Ala

2885 2890 2895

Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala

2900 2905 2910

Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys

2915 2920 2925

Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr

2930 2935 2940

Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys

2945 2950 2955

Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg

2960 2965 2970

Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp

2975 2980 2985

Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu

2990 2995 3000

His Lys Leu Gly Tyr Ile Leu Arg Glu Val Ser Lys Lys Glu Gly

3005 3010 3015

Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile

3020 3025 3030

Thr Ile Glu Asp Leu Lys Asn Glu Glu Met Ile Thr Asn His Met

3035 3040 3045

Ala Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr

3050 3055 3060

Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly

3065 3070 3075

Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly

3080 3085 3090

Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala

3095 3100 3105

Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Ile Phe Lys Ser Ile

3110 3115 3120

Gln His Leu Thr Ala Ser Glu Glu Ile Ala Val Gln Asp Trp Leu

3125 3130 3135

Val Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly

3140 3145 3150

Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Arg Ala

3155 3160 3165

Leu Thr Ala Leu Asn Asp Met Gly Lys Val Arg Lys Asp Ile Gln

3170 3175 3180

Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro

3185 3190 3195

Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg

3200 3205 3210

Thr Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg

3215 3220 3225

Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala

3230 3235 3240

Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe

3245 3250 3255

His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala

3260 3265 3270

Val Pro Ser His Trp Ile Pro Thr Ser Arg Thr Thr Trp Ser Ile

3275 3280 3285

His Ala Ser His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val

3290 3295 3300

Trp Asn Arg Val Trp Ile Leu Glu Asn Pro Trp Met Glu Asp Lys

3305 3310 3315

Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg

3320 3325 3330

Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala

3335 3340 3345

Thr Trp Ala Lys Asn Ile Gln Thr Ala Ile Asn Gln Val Arg Ser

3350 3355 3360

Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys

3365 3370 3375

Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp

3380 3385 3390

<210> SEQ ID NO: 32

<211> LENGTH: 15053

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Dengue 3 (Sleman/78) plasmid p3

<400> SEQENCE: 32

agttgttagt ctacgtggac cgacaagaac agtttcgact cggaagcttg cttaacgtag 60

tactgacagt tttttattag agagcagatc tctgatgaac aaccaacgga aaaagacggg 120

aaaaccgtct atcaatatgc tgaaacgcgt gagaaaccgt gtgtcaactg gatcacagtt 180

ggcgaagaga ttctcaagag gactgctgaa cggccaagga ccaatgaaat tggttatggc 240

gttcatagct ttcctcagat ttctagccat tccaccgaca gcaggagtct tggctagatg 300

gggaaccttt aagaagtcgg gggctattaa ggtcctgaga ggcttcaaga aggagatctc 360

aaacatgctg agcattatca acagacggaa aaagacatcg ctctgtctca tgatgatgtt 420

accagcaaca cttgctttcc acttgacttc acgagatgga gagccgcgca tgattgtggg 480

gaagaatgaa agaggaaaat ccctactttt taagacagcc tctggaatca acatgtgcac 540

actcatagcc atggatttgg gagagatgtg tgatgacacg gtcacctaca aatgccccct 600

cattactgaa gtggagcctg aagacattga ctgctggtgc aaccttacat cgacatgggt 660

gacctacgga acgtgcaatc aagctggaga gcacagacgc gacaaaagat cggtggcgtt 720

agctccccat gtcggcatgg gactggacac acgcacccaa acctggatgt cggctgaagg 780

agcttggaga caggtcgaga aggtagagac atgggccttt aggcacccag ggttcacaat 840

actagcccta tttcttgccc attacatagg cacttccttg acccagaaag tggttatttt 900

catactacta atgctggtca ccccatccat gacaatgaga tgcgtgggag taggaaacag 960

agattttgtg gaaggcctat caggagctac gtgggttgac gtggtgctcg agcacggtgg 1020

gtgtgtgact accatggcta agaacaagcc cacgctggat atagagctcc agaagaccga 1080

ggccacccaa ctggcgaccc taaggaaact atgtattgag ggaaaaatta ccaacgtaac 1140

aaccgactca aggtgcccca cccaagggga agcgatttta cctgaggagc aggaccagaa 1200

ccacgtgtgc aagcacacat acgtggacag aggctgggga aacggttgtg gtttgtttgg 1260

caagggaagc ctggtaacat gcgcgaaatt tcaatgtttg gaatcaatag agggaaaagt 1320

ggtgcagcat gagaacctca aatacaccgt catcatcaca gtgcacacag gagatcaaca 1380

ccaggtggga aatgaaacgc agggagtcac ggctgagata acaccccagg catcaaccgt 1440

tgaagccatc ttacctgaat atggaaccct tgggctagaa tgctcaccac ggacaggttt 1500

agatttcaat gaaatgattt tgttgacaat gaagaacaaa gcatggatgg tacatagaca 1560

atggtttttt gacctacctt taccatggac atcaggagct acaacagaaa caccaacctg 1620

gaataagaaa gagcttcttg tgacattcaa aaacgcacat gcaaaaaagc aagaagtagt 1680

agtccttgga tcgcaagagg gagcaatgca cacagcactg acaggagcta cagagatcca 1740

aacctcagga ggcacaagta tttttgcggg gcacttaaaa tgtagactca agatggacaa 1800

attggaactc aaggggatga gctatgcaat gtgcttgaat gcctttgtgt tgaagaaaga 1860

agtctccgaa acgcaacatg ggacaatact catcaaggtt gagtacaaag gggaagatgc 1920

accttgcaag attcctttct ccacggagga tggacaaggg aaagcccaca atggcagact 1980

gatcacagct aacccagtgg tgaccaagaa ggaggagcct gtcaatattg aggcagaacc 2040

tccttttggg gaaagcaata tagtaattgg aattggagac aaagccttga aaatcaactg 2100

gtacaagaag ggaagctcga ttgggaagat gttcgaggcc actgccagag gtgcaaggcg 2160

catggccatc ttgggagaca cagcctggga ctttggatca gtaggtggtg ttttaaattc 2220

attaggaaaa atggtgcacc aaatatttgg aagtgcttac acagccctat ttagtggagt 2280

ctcctggata atgaaaattg gaataggtgt ccttttaacc tggatagggt tgaattcaaa 2340

aaacactagt atgagcttta gctgcattgt gataggaatc attacactct atctgggagc 2400

cgtggtgcaa gctgacatgg ggtgtgtcat aaactggaaa ggcaaagaac tcaaatgtgg 2460

aagtggaatt ttcgtcacta atgaggtcca cacctggaca gagcaataca aatttcaagc 2520

agactccccc aaaagactgg cgacagccat tgcaggcgct tgggagaatg gagtgtgcgg 2580

aatcaggtcg acaaccagaa tggagaacct cttgtggaag caaatagcca atgaactgaa 2640

ctacatatta tgggaaaaca acatcaaatt aacggtagtt gtgggtgata taattggggt 2700

cttagagcaa gggaaaagaa cactaacacc acaacccatg gaactaaaat attcatggaa 2760

aacatgggga aaggcgaaga tagtgacagc tgaaacacaa aattcctctt tcataataga 2820

tgggccaaac acaccagagt gtccaagtgc ctcaagagca tggaatgtgt gggaggtgga 2880

agattacggg ttcggagtct tcacaactaa catatggctg aaactccgag agatgtacac 2940

ccaactatgt gaccacaggc taatgtcggc agccgttaag gatgagaggg ccgtacacgc 3000

cgacatgggc tattggatag aaagccaaaa gaatggaagt tggaagctag aaaaggcatc 3060

cctcatagag gtaaaaacct gcacatggcc aaaatcacac actctttgga gcaatggtgt 3120

gctagagagt gacatgatca tcccaaagag tctggctggt cccatttcgc aacacaacta 3180

caggcccgga taccacaccc aaacggcagg accctggcac ttaggaaaat tggagctgga 3240

cttcaactat tgtgaaggaa caacagttgt catcacagaa aattgtggga caagaggccc 3300

atcactgaga acaacaacag tgtcagggaa gttgatacac gaatggtgtt gccgctcgtg 3360

tacacttcct cccctgcgat acatgggaga agacggctgc tggtatggca tggaaattag 3420

acccattaat gagaaagaag agaacatggt aaagtcttta gtctcagcag ggagtggaaa 3480

ggtggataac ttcacaatgg gtgtcttgtg tttggcaatc ctttttgaag aggtgatgag 3540

aggaaaattt gggaaaaagc acatgattgc aggggttctc ttcacgtttg tactccttct 3600

ctcagggcaa ataacatgga gagacatggc gcacacactc ataatgattg ggtccaacgc 3660

ctctgacaga atgggaatgg gcgtcactta cctagcattg attgcaacat ttaaaattca 3720

gccatttttg gctttgggat tcttcctgag gaaactgaca tctagagaaa atttattgtt 3780

gggagttggg ttggccatgg caacaacgtt acaactgcca gaggacattg aacaaatggc 3840

gaatggaata gctttagggc tcatggctct taaattaata acacaatttg aaacatacca 3900

actatggacg gcattagtct ccctaatgtg ttcaaataca attttcacgt tgactgttgc 3960

ctggagaaca gccaccctga ttttggccgg aatttctctt ttgccagtgt gccagtcttc 4020

gagcatgagg aaaacagatt ggctcccaat ggctgtggca gctatgggag ttccacccct 4080

accacttttt attttcagtt tgaaagatac gctcaaaagg agaagctggc cactgaatga 4140

gggggtgatg gctgttggac ttgtgagtat tctagctagt tctctcctta ggaatgacgt 4200

gcccatggct ggaccattag tggctggggg cttgctgata gcgtgctacg tcataactgg 4260

cacgtcagca gacctcactg tagaaaaagc agcagatgtg acatgggagg aagaggctga 4320

gcaaacagga gtgtcccaca atttaatgat cacagttgat gacgatggaa caatgagaat 4380

aaaagatgat gagactgaga acatcttaac agtgcttttg aaaacagcat tactaatagt 4440

gtcaggcatt tttccatact ccatacccgc aacactgttg gtctggcaca cttggcaaaa 4500

gcaaacccaa agatccggtg tcctatggga cgttcccagc cccccagaga cacagaaagc 4560

agaactggaa gaaggggttt ataggatcaa gcagcaagga atttttggga aaacccaagt 4620

gggggttgga gtacaaaaag aaggagtttt ccacaccatg tggcacgtca caagaggagc 4680

agtgttgaca cacaatggga aaagactgga accaaactgg gctagcgtga aaaaagatct 4740

gatttcatac ggaggaggat ggaaattgag tgcacaatgg caaaaaggag aggaggtgca 4800

ggttattgcc gtagagcctg ggaagaaccc aaagaacttt caaaccatgc caggcatttt 4860

ccagacaaca acaggggaga taggagcgat tgcactggac ttcaagcctg gaacttcagg 4920

atctcccatc ataaacagag agggaaaggt actgggattg tatggcaatg gagtggtcac 4980

aaagaatggt ggctatgtca gtggaatagc acaaacaaat gcagaaccag acggaccgac 5040

accagagttg gaagaagaga tgttcaaaaa gcgaaatcta accataatgg atctccatcc 5100

cgggtcagga aagacgcgga aatatcttcc agctattgtt agagaggcaa tcaagagacg 5160

cttaaggact ctaattttgg caccaacaag ggtagttgca gctgagatgg aagaagcatt 5220

gaaagggctc ccaataaggt atcaaacaac tgcaacaaaa tctgaacaca cagggagaga 5280

gattgttgat ctaatgtgcc acgcaacgtt cacaatgcgt ttgctgtcac cagtcagggt 5340

tccaaactac aacttgataa taatggatga ggctcatttc acagacccag ccagtatagc 5400

ggctagaggg tacatatcaa ctcgtgtagg aatgggagag gcagccgcaa ttttcatgac 5460

agccacaccc cctggaacag ctgatgcctt tcctcagagc aacgctccaa ttcaagatga 5520

agaaagagac ataccagaac gctcatggaa ttcaggcaat gaatggatta ccgactttgc 5580

cgggaagacg gtgtggtttg tccctagcat caaagctgga aatgacatag caaactgctt 5640

gcggaaaaat ggaaaaaagg tcattcaact tagtaggaag acttttgaca cagaatatca 5700

aaagactaaa ctaaatgatt gggactttgt ggtgacaaca gacatttcag aaatgggagc 5760

caatttcaaa gcagacagag tgatcgaccc aagaagatgt ctcaagccag tgattttgac 5820

agacggaccc gagcgcgtga tcctggcggg accaatgcca gtcaccgtag cgagcgctgc 5880

gcaaaggaga gggagagttg gcaggaaccc acaaaaagaa aatgaccaat acatattcat 5940

gggccagccc ctcaataatg atgaagacca tgctcactgg acagaagcaa aaatgctgct 6000

agacaacatc aacacaccag aagggatcat accagctctc tttgaaccag aaagggagaa 6060

gtcagccgcc atagacggcg aataccgcct gaagggtgag tccaggaaga ccttcgtgga 6120

actcatgagg aggggtgacc tcccagtttg gctagcccat aaagtagcat cagaagggat 6180

caaatataca gatagaaagt ggtgttttga tggagaacgc aacaatcaaa ttttagagga 6240

gaatatggat gtggaaatct ggacaaagga aggagaaaag aaaaaattga gacctaggtg 6300

gcttgatgcc cgcacttatt cagatccctt agcgctcaag gaattcaagg actttgcggc 6360

tggtagaaag tcaattgccc ttgatcttgt gacagaaata ggaagagtgc cttcacactt 6420

agctcacaga acgagaaacg ccctggacaa tctggtgatg ttgcacacgt cagaacatgg 6480

cgggagggcc tacaggcatg cagtggagga actaccagaa acaatggaaa cactcttact 6540

cctgggactc atgatcctgt taacaggtgg agcaatgctt ttcttgatat caggtaaagg 6600

gattggaaag acttcaatag gactcatttg tgtagctgct tccagcggta tgttatggat 6660

ggctgatgtc ccactccaat ggatcgcgtc tgccatagtc ctggagtttt ttatgatggt 6720

gttacttata ccagaaccag aaaagcagag aactccccaa gacaatcaac tcgcatatgt 6780

cgtgataggc atactcacac tggctgcaat agtagcagcc aatgaaatgg gactgttgga 6840

aaccacaaag agagatttag gaatgtccaa agaaccaggt gttgtttctc caaccagcta 6900

tttggatgtg gacttgcacc cagcatcagc ctggacattg tacgctgtgg ccacaacagt 6960

aataacacca atgttgagac ataccataga gaattccaca gcaaatgtgt ccctggcagc 7020

tatagccaac caggcagtgg tcctgatggg tttagacaaa ggatggccga tatcgaaaat 7080

ggacttaggc gtgccactat tggcactggg ttgttattca caagtgaacc cactaactct 7140

cacagcggca gttctcctgc tagtcacgca ttatgctatt ataggtccag gattgcaggc 7200

aaaagccact cgtgaagctc aaaaaaggac agctgctgga ataatgaaga atccaacggt 7260

ggatgggata atgacaatag acctagatcc tgtaatatac gattcaaaat ttgaaaagca 7320

actaggacag gttatgctcc tggttctgtg tgcagttcaa cttttgttaa tgagaacatc 7380

atgggctttt tgtgaagctc taaccctagc cacaggacca ataacaacac tctgggaagg 7440

atcacctggg aagttctgga acaccacgat agctgtttcc atggcgaaca tctttagagg 7500

gagctattta gcaggagctg ggcttgcttt ttctatcatg aaatcagttg gaacaggaaa 7560

gagagggaca gggtcacagg gtgaaacctt gggagaaaag tggaaaaaga aattgaatca 7620

attaccccgg aaagagtttg acctttacaa gaaatccgga atcactgaag tggatagaac 7680

agaagccaaa gaagggttga aaagaggaga aataacacac catgccgtgt ccagaggcag 7740

cgcaaaactt caatggttcg tggagagaaa catggtcatc cccgaaggaa gagtcataga 7800

cttaggctgt ggaagaggag gctggtcata ttattgtgca ggactgaaaa aagttacaga 7860

agtgcgagga tacacaaaag gcggcccagg acatgaagaa acattggaga atcttcacca 7920

agcccaacag tggaagaaag cagaaccata agagtcttga agatggttga accatggcta 7980

aaaaataacc agttttgcat taaagtattg aacccttaca tgccaactgt gattgagcac 8040

ctagaaagac tacaaaggaa acatggagga atgcttgtga gaaatccact ctcacgaaac 8100

tccacgcacg aaatgtactg gatatctaat ggcacaggca atatcgtttc ttcagtcaac 8160

atggtatcca gattgctact taacagattc acaatgacac ataggagacc caccatagag 8220

aaagatgtgg atttaggagc ggggacccga catgtcaatg cggaaccaga aacacccaac 8280

atggatgtca ttggggaaag aataagaagg atcaaggagg agcatagttc aacatggcac 8340

tatgatgatg aaaatcctta taaaacgtgg gcttaccatg gatcctatga agttaaggcc 8400

acaggctcag cctcctccat gataaatgga gtcgtgaaac tcctcacgaa accatgggat 8460

gtggtgccca tggtgacaca gatggcaatg acggatacaa ccccattcgg ccagcaaagg 8520

gtttttaaag agaaagtgga caccaggaca cccagaccta tgccaggaac aagaaaggtt 8580

atggagatca cagcggaatg gctttggaga accctgggaa ggaacaaaag acccagatta 8640

tgtacgagag aggagttcac aaaaaaggtc agaaccaacg cagctatggg cgccgttttt 8700

acagaggaga accaatggga cagtgctaga gctgctgttg aggatgaaga attctggaaa 8760

ctcgtggaca gagaacgtga actccacaaa ttgggcaagt gtggaagctg cgtttacaac 8820

atgatgggca agagagagaa gaaacttgga gagtttggca aagcaaaagg cagtagagcc 8880

atatggtaca tgtggttggg agccagatac cttgagttcg aagcactcgg attcttaaat 8940

gaagaccatt ggttctcgcg tgaaaactct tacagtggag tagaaggaga aggactgcac 9000

aagctgggat acatcttaag agacatttcc aagatacccg gaggagctat gtatgctgat 9060

gacacagctg gttgggacac aagaataaca gaagatgacc tgcacaatga ggaaaaaatc 9120

acacagcaaa tggaccctga acacaggcag ttagcaaacg ctatattcaa gctcacatac 9180

caaaacaaag tggtcaaagt tcaacgacca actccaaagg gcacggtaat ggacatcata 9240

tctaggaaag accaaagagg cagtggacag gtgggaactt atggtctgaa tacattcacc 9300

aacatggaag cccagttaat cagacaaatg gaaggagaag gtgtgttgtc gaaggcagac 9360

ctcgagaacc ctcatctgct agagaagaaa gttacacaat ggttggaaac aaaaggagtg 9420

gagaggttaa aaagaatggc catcagcggg gatgattgcg tggtgaaacc aattgatgac 9480

aggttcgcca atgccctgct tgccctgaat gacatgggaa aagttaggaa ggacatacct 9540

caatggcagc catcaaaggg atggcatgat tggcaacagg tccctttctg ctcccaccac 9600

tttcatgaat tgatcatgaa agatggaaga aagttggtag ttccctgcag acctcaggat 9660

gaattaatcg ggagagcgag aatctctcaa ggagcaggat ggagccttag agaaactgca 9720

tgcctaggga aagcctacgc ccaaatgtgg actctcatgt actttcacag aagagatctt 9780

agactagcat ccaacgccat atgttcagca gtaccagtcc attgggtccc cacaagcaga 9840

acgacgtggt ctattcatgc tcaccatcag tggatgacta cagaagacat gcttactgtt 9900

tggaacaggg tgtggataga ggataatcca tggatggaag acaaaactcc agtcaaaacc 9960

tgggaagatg ttccatatct agggaagaga gaagaccaat ggtgcggatc actcattggt 10020

ctcacttcca gagcaacctg ggcccagaac atacttacgg caatccaaca ggtgagaagc 10080

cttataggca atgaagagtt tctggactac atgccttcga tgaagagatt caggaaggag 10140

gaggagtcag agggagccat ttggtaaacg taggaagtga aaaagaggca aactgtcagg 10200

ccaccttaag ccacagtacg gaagaagctg tgcagcctgt gagccccgtc caaggacgtt 10260

aaaagaagaa gtcaggccca aaagccacgg tttgagcaaa ccgtgctgcc tgtggctccg 10320

tcgtggggac gtaaaacctg ggaggctgca aactgtggaa gctgtacgca cggtgtagca 10380

gactagcggt tagaggagac ccctcccatg acacaacgca gcagcggggc ccgagctctg 10440

agggaagctg tacctccttg caaaggacta gaggttagag gagacccccc gcaaataaaa 10500

acagcatatt gacgctggga gagaccagag atcctgctgt ctcctcagca tcattccagg 10560

cacagaacgc cagaaaatgg aatggtgctg ttgaatcaac aggttctggt accggtaggc 10620

atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 10680

aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 10740

atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 10800

aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 10860

aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg 10920

gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 10980

gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 11040

gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 11100

ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 11160

ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 11220

atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 11280

gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 11340

atcacgaggc cctttcgtct tcaagaattc tcatgtttga cagcttatca tcgataagct 11400

ttaatgcggt agtttatcac agttaaattg ctaacgcagt caggcaccgt gtatgaaatc 11460

taacaatgcg ctcatcgtca tcctcggcac cgtcaccctg gatgctgtag gcataggctt 11520

ggttatgccg gtactgccgg gcctcttgcg ggatatcgtc cattccgaca gcatcgccag 11580

tcactatggc gtgctgctgg cgctatatgc gttgatgcaa tttctatgcg cacccgttct 11640

cggagcactg tccgaccgct ttggccgccg cccagtcctg ctcgcttcgc tacttggagc 11700

cactatcgac tacgcgatca tggcgaccac acccgtcctg tggatcctct acgccggacg 11760

catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat 11820

caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg 11880

tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt 11940

ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga 12000

gtcgcataag ggagagcgtc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt 12060

ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca 12120

actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg 12180

gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca 12240

agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg 12300

catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag gctggatggc 12360

cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt tgcaggccat 12420

gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc tcgcggctct 12480

taccagccta acttcgatca ctggaccgct gatcgtcacg gcgatttatg ccgcctcggc 12540

gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg tctgcctccc 12600

cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag ccggcggcac 12660

ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg gagaactgtg 12720

aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc cagcagccgc 12780

acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat cgtgctcctg 12840

tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg 12900

atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca 12960

tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc 13020

accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca 13080

tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc 13140

cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta 13200

acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat 13260

cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc 13320

tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa 13380

caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc 13440

gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 13500

gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 13560

ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 13620

ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 13680

cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 13740

actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 13800

tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 13860

aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 13920

ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 13980

aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 14040

cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 14100

cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 14160

aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 14220

cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 14280

ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 14340

ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 14400

gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 14460

agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 14520

acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 14580

tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 14640

agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 14700

gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 14760

agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 14820

cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 14880

ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 14940

cagttaatag tttgcgcaac gttgttgcca ttgctgcaag atctggctag cgatgaccct 15000

gctgattggt tcgctgacca tttccgggcg cgccgattta ggtgacacta tag 15053

<210> SEQ ID NO: 33

<211> LENGTH: 3390

<212> TYPE: PRT

<213> ORGANISM: Dengue 3 (Sleman/78)

<400> SEQENCE: 33

Met Asn Asn Gln Arg Lys Lys Thr Gly Lys Pro Ser Ile Asn Met Leu

1 5 10 15

Lys Arg Val Arg Asn Arg Val Ser Thr Gly Ser Gln Leu Ala Lys Arg

20 25 30

Phe Ser Arg Gly Leu Leu Asn Gly Gln Gly Pro Met Lys Leu Val Met

35 40 45

Ala Phe Ile Ala Phe Leu Arg Phe Leu Ala Ile Pro Pro Thr Ala Gly

50 55 60

Val Leu Ala Arg Trp Gly Thr Phe Lys Lys Ser Gly Ala Ile Lys Val

65 70 75 80

Leu Arg Gly Phe Lys Lys Glu Ile Ser Asn Met Leu Ser Ile Ile Asn

85 90 95

Arg Arg Lys Lys Thr Ser Leu Cys Leu Met Met Met Leu Pro Ala Thr

100 105 110

Leu Ala Phe His Leu Thr Ser Arg Asp Gly Glu Pro Arg Met Ile Val

115 120 125

Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala Ser Gly

130 135 140

Ile Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Asp

145 150 155 160

Asp Thr Val Thr Tyr Lys Cys Pro Leu Ile Thr Glu Val Glu Pro Glu

165 170 175

Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly

180 185 190

Thr Cys Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala

195 200 205

Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gln Thr Trp

210 215 220

Met Ser Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val Glu Thr Trp

225 230 235 240

Ala Phe Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe Leu Ala His

245 250 255

Tyr Ile Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe Ile Leu Leu

260 265 270

Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val Gly Asn

275 280 285

Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val

290 295 300

Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr

305 310 315 320

Leu Asp Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu Ala Thr Leu

325 330 335

Arg Lys Leu Cys Ile Glu Gly Lys Ile Thr Asn Val Thr Thr Asp Ser

340 345 350

Arg Cys Pro Thr Gln Gly Glu Ala Ile Leu Pro Glu Glu Gln Asp Gln

355 360 365

Asn His Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly Asn Gly

370 375 380

Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Gln

385 390 395 400

Cys Leu Glu Ser Ile Glu Gly Lys Val Val Gln His Glu Asn Leu Lys

405 410 415

Tyr Thr Val Ile Ile Thr Val His Thr Gly Asp Gln His Gln Val Gly

420 425 430

Asn Glu Thr Gln Gly Val Thr Ala Glu Ile Thr Pro Gln Ala Ser Thr

435 440 445

Val Glu Ala Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu Cys Ser

450 455 460

Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Ile Leu Leu Thr Met Lys

465 470 475 480

Asn Lys Ala Trp Met Val His Arg Gln Trp Phe Phe Asp Leu Pro Leu

485 490 495

Pro Trp Thr Ser Gly Ala Thr Thr Glu Thr Pro Thr Trp Asn Lys Lys

500 505 510

Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gln Glu Val

515 520 525

Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly

530 535 540

Ala Thr Glu Ile Gln Thr Ser Gly Gly Thr Ser Ile Phe Ala Gly His

545 550 555 560

Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly Met Ser

565 570 575

Tyr Ala Met Cys Leu Asn Ala Phe Val Leu Lys Lys Glu Val Ser Glu

580 585 590

Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp

595 600 605

Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala

610 615 620

His Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu

625 630 635 640

Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile

645 650 655

Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Lys Lys

660 665 670

Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg

675 680 685

Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly

690 695 700

Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gln Ile Phe Gly Ser

705 710 715 720

Ala Tyr Thr Ala Leu Phe Ser Gly Val Ser Trp Ile Met Lys Ile Gly

725 730 735

Ile Gly Val Leu Leu Thr Trp Ile Gly Leu Asn Ser Lys Asn Thr Ser

740 745 750

Met Ser Phe Ser Cys Ile Val Ile Gly Ile Ile Thr Leu Tyr Leu Gly

755 760 765

Ala Val Val Gln Ala Asp Met Gly Cys Val Ile Asn Trp Lys Gly Lys

770 775 780

Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Thr Asn Glu Val His Thr

785 790 795 800

Trp Thr Glu Gln Tyr Lys Phe Gln Ala Asp Ser Pro Lys Arg Leu Ala

805 810 815

Thr Ala Ile Ala Gly Ala Trp Glu Asn Gly Val Cys Gly Ile Arg Ser

820 825 830

Thr Thr Arg Met Glu Asn Leu Leu Trp Lys Gln Ile Ala Asn Glu Leu

835 840 845

Asn Tyr Ile Leu Trp Glu Asn Asn Ile Lys Leu Thr Val Val Val Gly

850 855 860

Asp Ile Ile Gly Val Leu Glu Gln Gly Lys Arg Thr Leu Thr Pro Gln

865 870 875 880

Pro Met Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Ile

885 890 895

Val Thr Ala Glu Thr Gln Asn Ser Ser Phe Ile Ile Asp Gly Pro Asn

900 905 910

Thr Pro Glu Cys Pro Ser Ala Ser Arg Ala Trp Asn Val Trp Glu Val

915 920 925

Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu

930 935 940

Arg Glu Met Tyr Thr Gln Leu Cys Asp His Arg Leu Met Ser Ala Ala

945 950 955 960

Val Lys Asp Glu Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu

965 970 975

Ser Gln Lys Asn Gly Ser Trp Lys Leu Glu Lys Ala Ser Leu Ile Glu

980 985 990

Val Lys Thr Cys Thr Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly

995 1000 1005

Val Leu Glu Ser Asp Met Ile Ile Pro Lys Ser Leu Ala Gly Pro

1010 1015 1020

Ile Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Thr Ala

1025 1030 1035

Gly Pro Trp His Leu Gly Lys Leu Glu Leu Asp Phe Asn Tyr Cys

1040 1045 1050

Glu Gly Thr Thr Val Val Ile Thr Glu Asn Cys Gly Thr Arg Gly

1055 1060 1065

Pro Ser Leu Arg Thr Thr Thr Val Ser Gly Lys Leu Ile His Glu

1070 1075 1080

Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Met Gly

1085 1090 1095

Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Ile Asn Glu

1100 1105 1110

Lys Glu Glu Asn Met Val Lys Ser Leu Val Ser Ala Gly Ser Gly

1115 1120 1125

Lys Val Asp Asn Phe Thr Met Gly Val Leu Cys Leu Ala Ile Leu

1130 1135 1140

Phe Glu Glu Val Met Arg Gly Lys Phe Gly Lys Lys His Met Ile

1145 1150 1155

Ala Gly Val Leu Phe Thr Phe Val Leu Leu Leu Ser Gly Gln Ile

1160 1165 1170

Thr Trp Arg Asp Met Ala His Thr Leu Ile Met Ile Gly Ser Asn

1175 1180 1185

Ala Ser Asp Arg Met Gly Met Gly Val Thr Tyr Leu Ala Leu Ile

1190 1195 1200

Ala Thr Phe Lys Ile Gln Pro Phe Leu Ala Leu Gly Phe Phe Leu

1205 1210 1215

Arg Lys Leu Thr Ser Arg Glu Asn Leu Leu Leu Gly Val Gly Leu

1220 1225 1230

Ala Met Ala Thr Thr Leu Gln Leu Pro Glu Asp Ile Glu Gln Met

1235 1240 1245

Ala Asn Gly Ile Ala Leu Gly Leu Met Ala Leu Lys Leu Ile Thr

1250 1255 1260

Gln Phe Glu Thr Tyr Gln Leu Trp Thr Ala Leu Val Ser Leu Met

1265 1270 1275

Cys Ser Asn Thr Ile Phe Thr Leu Thr Val Ala Trp Arg Thr Ala

1280 1285 1290

Thr Leu Ile Leu Ala Gly Ile Ser Leu Leu Pro Val Cys Gln Ser

1295 1300 1305

Ser Ser Met Arg Lys Thr Asp Trp Leu Pro Met Ala Val Ala Ala

1310 1315 1320

Met Gly Val Pro Pro Leu Pro Leu Phe Ile Phe Ser Leu Lys Asp

1325 1330 1335

Thr Leu Lys Arg Arg Ser Trp Pro Leu Asn Glu Gly Val Met Ala

1340 1345 1350

Val Gly Leu Val Ser Ile Leu Ala Ser Ser Leu Leu Arg Asn Asp

1355 1360 1365

Val Pro Met Ala Gly Pro Leu Val Ala Gly Gly Leu Leu Ile Ala

1370 1375 1380

Cys Tyr Val Ile Thr Gly Thr Ser Ala Asp Leu Thr Val Glu Lys

1385 1390 1395

Ala Ala Asp Val Thr Trp Glu Glu Glu Ala Glu Gln Thr Gly Val

1400 1405 1410

Ser His Asn Leu Met Ile Thr Val Asp Asp Asp Gly Thr Met Arg

1415 1420 1425

Ile Lys Asp Asp Glu Thr Glu Asn Ile Leu Thr Val Leu Leu Lys

1430 1435 1440

Thr Ala Leu Leu Ile Val Ser Gly Ile Phe Pro Tyr Ser Ile Pro

1445 1450 1455

Ala Thr Leu Leu Val Trp His Thr Trp Gln Lys Gln Thr Gln Arg

1460 1465 1470

Ser Gly Val Leu Trp Asp Val Pro Ser Pro Pro Glu Thr Gln Lys

1475 1480 1485

Ala Glu Leu Glu Glu Gly Val Tyr Arg Ile Lys Gln Gln Gly Ile

1490 1495 1500

Phe Gly Lys Thr Gln Val Gly Val Gly Val Gln Lys Glu Gly Val

1505 1510 1515

Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Thr His

1520 1525 1530

Asn Gly Lys Arg Leu Glu Pro Asn Trp Ala Ser Val Lys Lys Asp

1535 1540 1545

Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Ser Ala Gln Trp Gln

1550 1555 1560

Lys Gly Glu Glu Val Gln Val Ile Ala Val Glu Pro Gly Lys Asn

1565 1570 1575

Pro Lys Asn Phe Gln Thr Met Pro Gly Ile Phe Gln Thr Thr Thr

1580 1585 1590

Gly Glu Ile Gly Ala Ile Ala Leu Asp Phe Lys Pro Gly Thr Ser

1595 1600 1605

Gly Ser Pro Ile Ile Asn Arg Glu Gly Lys Val Leu Gly Leu Tyr

1610 1615 1620

Gly Asn Gly Val Val Thr Lys Asn Gly Gly Tyr Val Ser Gly Ile

1625 1630 1635

Ala Gln Thr Asn Ala Glu Pro Asp Gly Pro Thr Pro Glu Leu Glu

1640 1645 1650

Glu Glu Met Phe Lys Lys Arg Asn Leu Thr Ile Met Asp Leu His

1655 1660 1665

Pro Gly Ser Gly Lys Thr Arg Lys Tyr Leu Pro Ala Ile Val Arg

1670 1675 1680

Glu Ala Ile Lys Arg Arg Leu Arg Thr Leu Ile Leu Ala Pro Thr

1685 1690 1695

Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Lys Gly Leu Pro

1700 1705 1710

Ile Arg Tyr Gln Thr Thr Ala Thr Lys Ser Glu His Thr Gly Arg

1715 1720 1725

Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu

1730 1735 1740

Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp

1745 1750 1755

Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr

1760 1765 1770

Ile Ser Thr Arg Val Gly Met Gly Glu Ala Ala Ala Ile Phe Met

1775 1780 1785

Thr Ala Thr Pro Pro Gly Thr Ala Asp Ala Phe Pro Gln Ser Asn

1790 1795 1800

Ala Pro Ile Gln Asp Glu Glu Arg Asp Ile Pro Glu Arg Ser Trp

1805 1810 1815

Asn Ser Gly Asn Glu Trp Ile Thr Asp Phe Ala Gly Lys Thr Val

1820 1825 1830

Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Asn Cys

1835 1840 1845

Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys Thr

1850 1855 1860

Phe Asp Thr Glu Tyr Gln Lys Thr Lys Leu Asn Asp Trp Asp Phe

1865 1870 1875

Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys Ala

1880 1885 1890

Asp Arg Val Ile Asp Pro Arg Arg Cys Leu Lys Pro Val Ile Leu

1895 1900 1905

Thr Asp Gly Pro Glu Arg Val Ile Leu Ala Gly Pro Met Pro Val

1910 1915 1920

Thr Val Ala Ser Ala Ala Gln Arg Arg Gly Arg Val Gly Arg Asn

1925 1930 1935

Pro Gln Lys Glu Asn Asp Gln Tyr Ile Phe Met Gly Gln Pro Leu

1940 1945 1950

Asn Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu

1955 1960 1965

Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu Phe

1970 1975 1980

Glu Pro Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly Glu Tyr Arg

1985 1990 1995

Leu Lys Gly Glu Ser Arg Lys Thr Phe Val Glu Leu Met Arg Arg

2000 2005 2010

Gly Asp Leu Pro Val Trp Leu Ala His Lys Val Ala Ser Glu Gly

2015 2020 2025

Ile Lys Tyr Thr Asp Arg Lys Trp Cys Phe Asp Gly Glu Arg Asn

2030 2035 2040

Asn Gln Ile Leu Glu Glu Asn Met Asp Val Glu Ile Trp Thr Lys

2045 2050 2055

Glu Gly Glu Lys Lys Lys Leu Arg Pro Arg Trp Leu Asp Ala Arg

2060 2065 2070

Thr Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Asp Phe Ala

2075 2080 2085

Ala Gly Arg Lys Ser Ile Ala Leu Asp Leu Val Thr Glu Ile Gly

2090 2095 2100

Arg Val Pro Ser His Leu Ala His Arg Thr Arg Asn Ala Leu Asp

2105 2110 2115

Asn Leu Val Met Leu His Thr Ser Glu His Gly Gly Arg Ala Tyr

2120 2125 2130

Arg His Ala Val Glu Glu Leu Pro Glu Thr Met Glu Thr Leu Leu

2135 2140 2145

Leu Leu Gly Leu Met Ile Leu Leu Thr Gly Gly Ala Met Leu Phe

2150 2155 2160

Leu Ile Ser Gly Lys Gly Ile Gly Lys Thr Ser Ile Gly Leu Ile

2165 2170 2175

Cys Val Ala Ala Ser Ser Gly Met Leu Trp Met Ala Asp Val Pro

2180 2185 2190

Leu Gln Trp Ile Ala Ser Ala Ile Val Leu Glu Phe Phe Met Met

2195 2200 2205

Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp

2210 2215 2220

Asn Gln Leu Ala Tyr Val Val Ile Gly Ile Leu Thr Leu Ala Ala

2225 2230 2235

Ile Val Ala Ala Asn Glu Met Gly Leu Leu Glu Thr Thr Lys Arg

2240 2245 2250

Asp Leu Gly Met Ser Lys Glu Pro Gly Val Val Ser Pro Thr Ser

2255 2260 2265

Tyr Leu Asp Val Asp Leu His Pro Ala Ser Ala Trp Thr Leu Tyr

2270 2275 2280

Ala Val Ala Thr Thr Val Ile Thr Pro Met Leu Arg His Thr Ile

2285 2290 2295

Glu Asn Ser Thr Ala Asn Val Ser Leu Ala Ala Ile Ala Asn Gln

2300 2305 2310

Ala Val Val Leu Met Gly Leu Asp Lys Gly Trp Pro Ile Ser Lys

2315 2320 2325

Met Asp Leu Gly Val Pro Leu Leu Ala Leu Gly Cys Tyr Ser Gln

2330 2335 2340

Val Asn Pro Leu Thr Leu Thr Ala Ala Val Leu Leu Leu Val Thr

2345 2350 2355

His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr Arg

2360 2365 2370

Glu Ala Gln Lys Arg Thr Ala Ala Gly Ile Met Lys Asn Pro Thr

2375 2380 2385

Val Asp Gly Ile Met Thr Ile Asp Leu Asp Pro Val Ile Tyr Asp

2390 2395 2400

Ser Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val Leu

2405 2410 2415

Cys Ala Val Gln Leu Leu Leu Met Arg Thr Ser Trp Ala Phe Cys

2420 2425 2430

Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Thr Thr Leu Trp Glu

2435 2440 2445

Gly Ser Pro Gly Lys Phe Trp Asn Thr Thr Ile Ala Val Ser Met

2450 2455 2460

Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala

2465 2470 2475

Phe Ser Ile Met Lys Ser Val Gly Thr Gly Lys Arg Gly Thr Gly

2480 2485 2490

Ser Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys Lys Lys Leu Asn

2495 2500 2505

Gln Leu Pro Arg Lys Glu Phe Asp Leu Tyr Lys Lys Ser Gly Ile

2510 2515 2520

Thr Glu Val Asp Arg Thr Glu Ala Lys Glu Gly Leu Lys Arg Gly

2525 2530 2535

Glu Ile Thr His His Ala Val Ser Arg Gly Ser Ala Lys Leu Gln

2540 2545 2550

Trp Phe Val Glu Arg Asn Met Val Ile Pro Glu Gly Arg Val Ile

2555 2560 2565

Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly

2570 2575 2580

Leu Lys Lys Val Thr Glu Val Arg Gly Tyr Thr Lys Gly Gly Pro

2585 2590 2595

Gly His Glu Glu Pro Val Pro Met Ser Thr Tyr Gly Trp Asn Ile

2600 2605 2610

Val Lys Leu Met Ser Gly Lys Asp Val Phe Tyr Leu Pro Pro Glu

2615 2620 2625

Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro Ser

2630 2635 2640

Pro Thr Val Glu Glu Ser Arg Thr Ile Arg Val Leu Lys Met Val

2645 2650 2655

Glu Pro Trp Leu Lys Asn Asn Gln Phe Cys Ile Lys Val Leu Asn

2660 2665 2670

Pro Tyr Met Pro Thr Val Ile Glu His Leu Glu Arg Leu Gln Arg

2675 2680 2685

Lys His Gly Gly Met Leu Val Arg Asn Pro Leu Ser Arg Asn Ser

2690 2695 2700

Thr His Glu Met Tyr Trp Ile Ser Asn Gly Thr Gly Asn Ile Val

2705 2710 2715

Ser Ser Val Asn Met Val Ser Arg Leu Leu Leu Asn Arg Phe Thr

2720 2725 2730

Met Thr His Arg Arg Pro Thr Ile Glu Lys Asp Val Asp Leu Gly

2735 2740 2745

Ala Gly Thr Arg His Val Asn Ala Glu Pro Glu Thr Pro Asn Met

2750 2755 2760

Asp Val Ile Gly Glu Arg Ile Arg Arg Ile Lys Glu Glu His Ser

2765 2770 2775

Ser Thr Trp His Tyr Asp Asp Glu Asn Pro Tyr Lys Thr Trp Ala

2780 2785 2790

Tyr His Gly Ser Tyr Glu Val Lys Ala Thr Gly Ser Ala Ser Ser

2795 2800 2805

Met Ile Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Val

2810 2815 2820

Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe

2825 2830 2835

Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro

2840 2845 2850

Arg Pro Met Pro Gly Thr Arg Lys Val Met Glu Ile Thr Ala Glu

2855 2860 2865

Trp Leu Trp Arg Thr Leu Gly Arg Asn Lys Arg Pro Arg Leu Cys

2870 2875 2880

Thr Arg Glu Glu Phe Thr Lys Lys Val Arg Thr Asn Ala Ala Met

2885 2890 2895

Gly Ala Val Phe Thr Glu Glu Asn Gln Trp Asp Ser Ala Arg Ala

2900 2905 2910

Ala Val Glu Asp Glu Glu Phe Trp Lys Leu Val Asp Arg Glu Arg

2915 2920 2925

Glu Leu His Lys Leu Gly Lys Cys Gly Ser Cys Val Tyr Asn Met

2930 2935 2940

Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys

2945 2950 2955

Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Tyr Leu

2960 2965 2970

Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser

2975 2980 2985

Arg Glu Asn Ser Tyr Ser Gly Val Glu Gly Glu Gly Leu His Lys

2990 2995 3000

Leu Gly Tyr Ile Leu Arg Asp Ile Ser Lys Ile Pro Gly Gly Ala

3005 3010 3015

Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu

3020 3025 3030

Asp Asp Leu His Asn Glu Glu Lys Ile Thr Gln Gln Met Asp Pro

3035 3040 3045

Glu His Arg Gln Leu Ala Asn Ala Ile Phe Lys Leu Thr Tyr Gln

3050 3055 3060

Asn Lys Val Val Lys Val Gln Arg Pro Thr Pro Lys Gly Thr Val

3065 3070 3075

Met Asp Ile Ile Ser Arg Lys Asp Gln Arg Gly Ser Gly Gln Val

3080 3085 3090

Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu

3095 3100 3105

Ile Arg Gln Met Glu Gly Glu Gly Val Leu Ser Lys Ala Asp Leu

3110 3115 3120

Glu Asn Pro His Leu Leu Glu Lys Lys Val Thr Gln Trp Leu Glu

3125 3130 3135

Thr Lys Gly Val Glu Arg Leu Lys Arg Met Ala Ile Ser Gly Asp

3140 3145 3150

Asp Cys Val Val Lys Pro Ile Asp Asp Arg Phe Ala Asn Ala Leu

3155 3160 3165

Leu Ala Leu Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln

3170 3175 3180

Trp Gln Pro Ser Lys Gly Trp His Asp Trp Gln Gln Val Pro Phe

3185 3190 3195

Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Lys

3200 3205 3210

Leu Val Val Pro Cys Arg Pro Gln Asp Glu Leu Ile Gly Arg Ala

3215 3220 3225

Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys

3230 3235 3240

Leu Gly Lys Ala Tyr Ala Gln Met Trp Thr Leu Met Tyr Phe His

3245 3250 3255

Arg Arg Asp Leu Arg Leu Ala Ser Asn Ala Ile Cys Ser Ala Val

3260 3265 3270

Pro Val His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His

3275 3280 3285

Ala His His Gln Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp

3290 3295 3300

Asn Arg Val Trp Ile Glu Asp Asn Pro Trp Met Glu Asp Lys Thr

3305 3310 3315

Pro Val Lys Thr Trp Glu Asp Val Pro Tyr Leu Gly Lys Arg Glu

3320 3325 3330

Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr

3335 3340 3345

Trp Ala Gln Asn Ile Leu Thr Ala Ile Gln Gln Val Arg Ser Leu

3350 3355 3360

Ile Gly Asn Glu Glu Phe Leu Asp Tyr Met Pro Ser Met Lys Arg

3365 3370 3375

Phe Arg Lys Glu Glu Glu Ser Glu Gly Ala Ile Trp

3380 3385 3390

<210> SEQ ID NO: 34

<211> LENGTH: 51

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Forward primer

<400> SEQENCE: 34

tcaaaacaaa agaaaagatc tgcagtgacc ggaattgcag tcatgattgg c 51

<210> SEQ ID NO: 35

<211> LENGTH: 51

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Forward primer

<400> SEQENCE: 35

tcaaaacaaa agaaaagatc tgcagggacc ggaattgcag tcatgattgg c 51

<210> SEQ ID NO: 36

<211> LENGTH: 51

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Forward primer

<400> SEQENCE: 36

tcaaaacaaa agaaaagatc tgcagacacc ggaattgcag tcatgattgg c 51

<210> SEQ ID NO: 37

<211> LENGTH: 64

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<223> OTHER INFORMATION: Reverse primer

<400> SEQENCE: 37

ccgcaagaaa cgtcatagca attgacctgt cactcgagtt gattcccatc cacaacagaa 60

gagc 64

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Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
Attenuated dengue-4 virus vaccine WALTER REED ARMY INSTITUTE OF RESEARCH,ECKELS, KENNETH, H.,PUTNAK, JOSEPH, R.,DUBOIS, DORIA, R.,INNIS, BRUCE, L. 24 March 2000 05 October 2000
Chimeric and/or growth-restricted flaviviruses THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES,LAI, CHING-JUH,BRAY, MICHAEL,PLETNEV, ALEXANDER, G.,MEN, RUHE 18 September 1992 01 April 1993
Avirulent, immunogenic flavivirus chimeras DEPARTMENT OF HEALTH AND HUMAN SERVICES, CENTERS FOR DISEASE CONTROL AND PREVENTION, AS REPRESENTED BY GOVERMENT OF THE UNITED STATES OF AMERICA, THE,MAHIDOL UNIVERSITY 16 February 2001 22 August 2006
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The widest range of IP search tools makes getting the right answers and asking the right questions easier than ever. One click analysis extracts meaningful information on competitors and technology trends from IP data.
Business Intelligence
Gain powerful insights into future technology changes, market shifts and competitor strategies.
Workflow
Manage IP-related processes across multiple teams and departments with integrated collaboration and workflow tools.
Contact Sales
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