U.S. patent number RE45,123 [Application Number 13/896,409] was granted by the patent office on 2014-09-09 for recombinant attenuated dengue viruses comprising a deletion in the 3' untranslated region and additional attenuating mutations induced by chemical mutagenesis.
This patent grant is currently assigned to N/A, The United States of America, as Represented by the Secretary, Department of Health and Human Services. The grantee listed for this patent is Government of The United States of America, as represented by the Secretary, Department of Health & Human Services, N/A, Government of The United States of America, as represented by the Secretary, Department of Health & Human Services. Invention is credited to Joseph E. Blaney, Kathryn A. Hanley, Ching-Juh Lai, Brian R. Murphy, Stephen S. Whitehead.
United States Patent |
RE45,123 |
Whitehead , et al. |
September 9, 2014 |
Recombinant attenuated dengue viruses comprising a deletion in the
3' untranslated region and additional attenuating mutations induced
by chemical mutagenesis
Abstract
The invention provides compositions featuring an attenuated
dengue virus mutant or an attenuated chimeric dengue virus
mutant.
Inventors: |
Whitehead; Stephen S.
(Montgomery Village, MD), Murphy; Brian R. (Bethesda,
MD), Hanley; Kathryn A. (Bethesda, MD), Blaney; Joseph
E. (Gettysburg, PA), Lai; Ching-Juh (Bethesda, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
Government of The United States of America, as represented by the
Secretary, Department of Health & Human Services
N/A |
Rockville |
MD |
US |
|
|
Assignee: |
The United States of America, as
Represented by the Secretary, Department of Health and Human
Services (Washington, DC)
N/A (N/A)
|
Family
ID: |
23127420 |
Appl.
No.: |
13/896,409 |
Filed: |
May 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11446050 |
Jul 14, 2009 |
7560118 |
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10719547 |
Jun 5, 2007 |
7226602 |
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PCT/US02/16308 |
May 22, 2002 |
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60293049 |
May 22, 2001 |
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Reissue of: |
12396376 |
Mar 2, 2009 |
8039003 |
Oct 18, 2011 |
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Current U.S.
Class: |
424/218.1;
435/236 |
Current CPC
Class: |
A61K
39/295 (20130101); A61K 39/12 (20130101); C12Q
1/701 (20130101); A61P 37/04 (20180101); A61P
31/14 (20180101); C12N 7/00 (20130101); C12N
7/04 (20130101); C12Q 2600/156 (20130101); C12N
2770/24151 (20130101); A61K 2039/70 (20130101); A61K
2039/5254 (20130101); Y02A 50/30 (20180101); A61K
2039/575 (20130101); C12N 2770/24134 (20130101); C12N
2770/24162 (20130101); C12N 2770/24121 (20130101); C12N
2770/24171 (20130101); A61K 2039/5256 (20130101); A61K
2039/54 (20130101); G01N 2333/18 (20130101); G01N
2500/10 (20130101); A61K 2039/5252 (20130101); C12N
2770/24161 (20130101) |
Current International
Class: |
A61K
39/12 (20060101); C12N 7/04 (20060101) |
Other References
R Men et al., "Dengue Type 4 Virus Mutants Containing Deletions in
the 3' Noncoding Region of the RNA Genome: Analysis of Growth
Restriction in Cell Culture and Altered Viremia Pattern and
Immunogenicity in Rhesus Monkeys", Journal of Virology, 70(6), pp.
3930-3937 (1996). cited by applicant .
M. Bray et al., "Monkeys Immunized with Interyptic Chimeric Dengue
Viruses Are Protected against Wild-Type Virus Challenge", Journal
of Virology, 70(6), pp. 4162-4166 (1996). cited by applicant .
European Search Report from European Patent Application No.
10181776, search completed on Jan. 28, 2011. cited by applicant
.
European Search Report from European Patent Application No.
10181776, search completed on Feb. 3, 2011. cited by applicant
.
European Search Report from European Patent Application No.
10181776, search completed on Feb. 11, 2011. cited by applicant
.
Men, R., et al., 1996, Dengue type 4 Virus mutants containing
deletions in the 3' noncoding region of the RNA genome: analysis of
growth restriction in cell culture and altered viremia pattern and
immunogenicity in rhesus monkeys, J. Virol. 70(6): 3930-3937. cited
by examiner .
Bray, M., et al., 1996, Monkeys immunized with interyptic chimeric
dengue viruses are protected against wild-type virus challenge, J.
Virol. 70(6):4162-4166. cited by examiner.
|
Primary Examiner: Parkin; Jeffrey S.
Attorney, Agent or Firm: Edwards Wildman Palmer LLP Corless;
Peter F. Emmons; Richard B.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
11/446,050, filed Jun. 2, 2006, now U.S. Pat. No. 7,560,118, which
is a divisional of U.S. application Ser. No. 10/719,547, filed Nov.
21, 2003, now U.S. Pat. No. 7,226,602, which is a continuation and
claims the benefit of priority of International Application No.
PCT/US02/16308 filed May 22, 2002, designating the United States of
America and published in English as WO 02/095075 on Nov. 28, 2002,
which claims the benefit of priority of U.S. Provisional
Application No. 60/293,049, filed May 22, 2001, all of which are
hereby expressly incorporated by reference in their entirety.
Claims
What is claimed is:
1. An immunogenic composition comprising an attenuated dengue virus
or attenuated chimeric dengue virus comprising (a) a nucleic acid
encoding a structural protein from dengue serotype 1, dengue
serotype 2, dengue serotype 3, or dengue serotype 4; and (b) a
nucleic acid comprising remaining coding and non-coding regions of
a dengue serotype 4 virus (DEN4 backbone), wherein the DEN4
backbone comprises a 30 nucleotide deletion in the 3' untranslated
region corresponding to nucleotides 172-143 (DEN4.DELTA.30
backbone), wherein the numbering is based upon a prototypic DEN4
Dominica 1981 isolate, and wherein the DEN4 backbone further
comprises one or more mutations from Table 37.
2. The composition of claim 1, wherein the nucleic acid encoding
the structural protein is from the dengue serotype 4 virus
(DEN4.DELTA.30).
3. The composition of claim 2, further comprising a first
attenuated chimeric virus having a nucleic acid encoding a
structural protein from dengue serotype 1 virus and a DEN4.DELTA.30
backbone (DEN1/4.DELTA.30).
4. The composition of claim 3, further comprising a second
attenuated chimeric virus comprising a nucleic acid encoding a
structural protein from dengue serotype 2 virus and a DEN4.DELTA.30
backbone (DEN2/4.DELTA.30).
5. The composition of claim 4, further comprising a third
attenuated chimeric virus comprising a nucleic acid encoding a
structural protein from dengue serotype 3 virus and a DEN4.DELTA.30
backbone (DEN3/4.DELTA.30).
6. The composition of claim 5, wherein a nucleic acid encoding the
NS3 protein of at least one of DEN4.DELTA.30, the first attenuated
chimeric virus, the second attenuated chimeric virus, and the third
attenuated chimeric virus comprises a mutation at nucleotide
position 4995 from T to C.
7. The composition of claim 1, wherein the nucleic acid encoding
the structural protein is from the dengue serotype 1 virus.
8. The composition of claim 1, wherein the nucleic acid encoding
the structural protein is from the dengue serotype 2 virus.
9. The composition of claim 1, wherein the nucleic acid encoding
the structural protein is from the dengue serotype 3 virus.
10. The composition of claim 1, wherein the mutation in the DEN4
backbone is a mutation at nucleotide position 4995 from T to C.
Description
SEQUENCE LISTING
The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled NIH214-1CDV1C1_Sequence Listing.TXT, created Feb. 20,
2009, which is 249 Kb in size. The information in the electronic
format of the Sequence Listing is incorporated herein by reference
in its entirety.
FIELD OF THE INVENTION
A menu of mutations was developed that is useful in fine-tuning the
attenuation and growth characteristics of dengue virus
vaccines.
BACKGROUND OF THE INVENTION
Dengue virus is a positive-sense RNA virus belonging to the
Flavivirus genus of the family Flaviviridae. Dengue virus is widely
distributed throughout the tropical and semitropical regions of the
world and is transmitted to humans by mosquito vectors. Dengue
virus is a leading cause of hospitalization and death in children
in at least eight tropical Asian countries (WHO, 1997. Dengue
haemorrhagic fever: diagnosis, treatment prevention and
control--2nd ed. Geneva: WHO). There are four serotypes of dengue
virus (DEN-1, DEN-2, DEN-3, and DEN-4) which annually cause an
estimated 50-100 million cases of dengue fever and 500,000 cases of
the more severe form of dengue virus infection, dengue hemorrhagic
fever/dengue shock syndrome (DHF/DSS) (Gubler, D. J. & Meltzer,
M. 1999 Adv Virus Res 53:35-70). DHF/DSS is seen predominately in
children and adults experiencing a second dengue virus infection
with a serotype different than that of their first dengue virus
infection and in primary infection of infants who still have
circulating dengue-specific maternal antibody (Burke, D. S. et al.
1988 Am J Trop Med Hyg 38:172-80; Halstead, S. B. et al. 1969 Am J
Trop Med Hyg 18:997-1021; Thein S. et al. 1997 Am J Trop Med Hyg
56:566-72). A vaccine is needed to lessen the disease burden caused
by dengue virus, but none is licensed. Because of the association
of more severe disease with secondary dengue virus infection, a
successful vaccine must induce immunity to all four serotypes.
Immunity is primarily mediated by neutralizing antibody directed
against the envelope E glycoprotein, a virion structural protein.
Infection with one serotype induces long-lived homotypic immunity
and a short-lived heterotypic immunity (Sabin, A. 1955 Amer J Trop
Med Hyg 4:198-207). Therefore, the goal of immunization is to
induce a long-lived neutralizing antibody response against DEN-1,
DEN-2, DEN-3, and DEN-4, which can best be achieved economically
using live attenuated virus vaccines. This is a reasonable goal
since a live attenuated vaccine has already been developed for the
related yellow fever virus, another mosquito-borne flavivirus
present in tropical and semitropical regions of the world (Monath,
T. P. & Heinz, F. X. 1996 in: Fields B. N. et al. eds. Fields
Virology Philadelphia: Lippincott-Ravan Publishers, 961-1034).
Several live attenuated dengue vaccine candidates have been
developed and evaluated in humans or non-human primates. The first
live attenuated dengue vaccine candidates were host range mutants
developed by serial passage of wild type dengue viruses in the
brains of mice and selection of mutants attenuated for humans
(Kimura, R. & Hotta, S. 1944 Japanese J Bacteriology 1:96-99;
Sabin, A. B. & Schlesinger, R. W. 1945 Science 101:640;
Wisseman, C. L. Jr. et al. 1963 Am J Trop Med 12:620-623). Although
these candidate vaccine viruses were immunogenic in humans, their
poor growth in cell culture discouraged further development.
Additional live attenuated DEN-1, DEN-2, DEN-3, and DEN-4 vaccine
candidates have been developed by serial passage in tissue culture
(Angsubhakorn, S. et al. 1994 Southeast Asian J Trop Med Public
Health 25:554-9; Bancroft, W. H. et al. 1981 Infect Immun
31:698-703; Bhamarapravati, N. et al. 1987 Bull World Health Organ
65:189-95; Eckels, K. H. et al. 1984 Am J Trop Med Hyg 33:684-9;
Hoke, C. H. Jr. et al. 1990 Am J Trop Med Hyg 43:219-26;
Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-88) or by chemical
mutagenesis (McKee, K. T. Jr. et al. 1987 Am J Trop Med Hyg
36:435-42). It has proven very difficult to achieve a satisfactory
balance between attenuation and immunogenicity for each of the four
serotypes of dengue virus using these approaches and to formulate a
tetravalent vaccine that is safe and satisfactorily immunogenic
against each of the four dengue viruses (Kanesathasan, N. et al.
2001 Vaccine 19:3179-88; Bhamarapravati, N. & Sutee, Y. 2000
Vaccine 18 Suppl 2: 44-7).
Two major advances utilizing recombinant DNA technology have
recently made it possible to develop additional promising live
attenuated dengue virus vaccine candidates. First, methods have
been developed to recover infectious dengue virus from cells
transfected with RNA transcripts derived from a full-length cDNA
clone of the dengue virus genome, thus making it possible to derive
infectious viruses bearing attenuating mutations which have been
introduced into the cDNA clone by site-directed mutagenesis (Lai,
C. J. et al. 1991 PNAS USA 88:5139-43). Second, it is possible to
produce antigenic chimeric viruses in which the structural protein
coding region of the full-length cDNA clone of dengue virus is
replaced by that of a different dengue virus serotype or from a
more divergent flavivirus (Bray, M. & Lai, C. J. 1991 PNAS USA
88: 10342-6; Chen, W. et al. 1995 J Virol 69:5186-90; Huang, C. Y
et al. 2000 J Virol 74:3020-8; Pletnev, A. G. & Men, R. 1998
PNAS USA 95:1746-51). These techniques have been used to construct
intertypic chimeric dengue viruses which have been shown to be
effective in protecting monkeys against homologous dengue virus
challenge (Bray, M. et al. 1996 J Virol 70:4162-6). Despite these
advances, there is a need to develop attenuated antigenic dengue
virus vaccines that specify a satisfactory balance between
attenuation and immunogenicity for humans.
SUMMARY OF THE INVENTION
The invention provides mutations that confer temperature
sensitivity in Vero cells or human liver cells, host-cell
restriction in mosquito or human liver cells, host-cell adaptation
for improved replication in Vero cells, or attenuation in mice,
which mutations are useful in fine tuning the attenuation and
growth characteristics of dengue virus vaccines.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows growth of wt DEN4 2A and vaccine candidate,
2A.DELTA.30, in Vero and HuH-7 cells. Vero (A) or HuH-7 (B) cells
were infected with DEN4 2A or 2A.DELTA.30 at a multiplicity of
infection (MOI) of 10 or 0.01. Confluent cell monolayers in 25-mm
tissue culture flasks were washed and overlaid with a 1.5 ml
inoculum containing the indicated virus. After a two hour
incubation at 37.degree. C., cells were washed three times in PBS
and 7 ml of culture media supplemented with 2% FBS was added. A 1
ml aliquot of tissue culture medium was removed, replaced with
fresh medium, and designated the 0 hour time-point. At the
indicated time points post-infection, samples of tissue culture
media were removed and frozen at -70.degree. C. The level of viral
replication was assayed by plaque titration in Vero cells. Briefly,
serial ten-fold dilutions of cell culture media samples were
inoculated onto confluent Vero cell monolayers in 24-well plates in
duplicate and overlaid with Opti-MEM containing 0.8%
methylcellulose. After five days, plaques were visualized by
immunoperoxidase staining as described in Example 1.
FIG. 2 shows generation of temperature-sensitive (ts) DEN4 viruses
by 5-fluorouracil (5-FU) chemical mutagenesis. The wild-type DEN4
2A virus was derived from a cDNA clone of DEN4 strain 814669
(Dominica, 1981). Vero cells were infected with DEN4 2A and
overlaid with culture media containing 1 mM 5-fluorouracil (5-FU)
which resulted in a reduction of approximately 100-fold in viral
replication when compared to untreated controls. Viral progeny from
the 1 mM 5-FU-treated cultures were subjected to a single round of
terminal dilutions generating 1,248 biologically cloned viruses
which were screened for ts phenotypes by assessing virus
replication at 35.degree. C. and 39.degree. C. in Vero and HuH-7
cells. Virus clones which demonstrated a 100-fold or greater
reduction in titer at 39.degree. C. were terminally diluted an
additional two times and amplified in Vero cells.
Temperature-sensitive phenotypes of the 3.times. biologically
cloned viruses were confirmed by evaluating efficiency of plaque
formation (EOP) in the indicated cells as described in Example
1.
FIG. 3 shows plaque size phenotypes of representative 5-FU mutant
DEN4 viruses. Serial ten-fold dilutions of wild-type DEN4 2A-13
(A), 5-FU mutant viruses #569 and #1189 (B), and 5-FU mutant
viruses #1083 and #311 (C) were inoculated onto confluent Vero and
HuH-7 cell monolayers in 24-well plates. After incubation at
35.degree. C. for two hours, monolayers were overlaid with 0.8%
methylcellulose culture media. Following incubation at 35.degree.
C. for five days, plaques were visualized by immunoperoxidase
staining. Viruses which had a plaque size that was .ltoreq.1 mm
(approximately .ltoreq.50% the size of wt DEN4 2A-13) at the
permissive temperature of 35.degree. C. were designated as having
the small-plaque (sp) phenotype. Mutant viruses #569 and #1189 (B)
were sp in both Vero and HuH-7 cells, and #311 and #1083 (C) were
sp in only HuH-7 cells.
FIG. 4 shows generation of recombinant DEN4 viruses. (A), The p4
cDNA clone is represented which was constructed from the 2A cDNA
clone (derived from DEN4 814669) by site-directed mutagenesis.
Restriction enzyme sites were introduced or removed to facilitate
subsequent cloning of DEN4 recombinants bearing introduced
attenuating mutations. Restriction enzyme sites are shown and
define fragments of the genome that were sub-cloned into modified
pUC-119 vectors for site-directed mutagenesis to introduce
mutations identified in the 5-FU mutant viruses. (B), An outline of
the methods used to generate rDEN4 viruses is also represented and
described in Example 1.
FIG. 5 shows amino acid sequence of the rDEN4 NS5 gene (SEQ ID NO:
1). Eighty underlined amino acid pairs were mutagenized to alanine
pairs; 32 pairs in boldface represent mutant viruses that could be
recovered in either Vero or C6/36 cells; pairs in normal type
represent mutant viruses that could not be recovered in either Vero
or C6/36 cells. Boxed regions indicate putative functional domains,
including an S-adenosylmethionine utilizing methyltransferase
domain (SAM), an importin-.beta. binding domain adjacent to a
nuclear localization sequence (importin-.beta.-binding+NLS) and an
RNA-dependent RNA polymerase domain (Polymerase).
FIG. 6 shows plaque size of mutant 5-1A1 in C6/36 cells. Note that
5-1A1 has a small plaque phenotype in C6/36 cells relative to that
of the wild type virus.
FIG. 7 shows growth of wild type rDEN4 and 5-1A1 in C6/36 cells.
Cells were inoculated in triplicate with each virus at an MOI of
0.01, and the amount of virus present in the supernatants that were
harvested on the indicated days was determined by plaque
enumeration in Vero cells. The titers are expressed as
log.sub.10PFU/ml.+-.standard error.
FIG. 8 shows nucleotide alignment of the 3' UTR of mosquito-borne
and tick-borne flaviviruses. cDNA sequences are shown 5' to 3' and
represent a portion of the UTR corresponding to DEN4 nucleotides
10417 to 10649 (3' genome end). Nucleotide numbering represents the
position in the alignment. Regions deleted or swapped are indicated
using the nucleotide numbering of DEN4. GenBank accession numbers
for mosquito-borne viruses: DEN4 (SEQ ID NO: 2): AF326825, DEN1
(SEQ ID NO: 3): U88535, DEN2 (SEQ ID NO: 4): AF038403, DEN3 (SEQ ID
NO: 5): M93130, West Nile virus (WN) (SEQ ID NO: 6): M12294,
Japanese encephalitis virus (JE) (SEQ ID NO: 7): AF315119, Yellow
fever virus (YF) (SEQ ID NO: 8): U17067; GenBank accession numbers
for tick-borne viruses: Powassan virus (POW) (SEQ ID NO: 9):
L06436, Louping Ill virus (LI) (SEQ ID NO: 10): Y07863, Tick-borne
encephalitis virus (TBE) (SEQ ID NO: 11): U27495, and Langat virus
(LGT) (SEQ ID NO: 12): AF253419.
FIG. 9 shows genetic map of plasmid p4. Dengue cDNA is shown as
bold line, with the C-prM-E region exchanged during construction of
chimeric dengue virus cDNAs indicated.
FIG. 10 shows plaque size phenotypes of rDEN4 viruses encoding Vero
adaptation mutations. Serial three-fold dilutions of the indicated
viruses were inoculated onto confluent Vero and C6/36 cell
monolayers in 6-well plates. After incubation at 37.degree. C.
(Vero) or 32.degree. C. (C6/36) for two hours, monolayers were
overlaid with 0.8% methylcellulose culture media. Following
incubation for five days, plaques were visualized by
immunoperoxidase staining. Values below each well are the average
plaque size in mm.+-.standard error. For each of the virus-infected
wells, 36 plaques were measured on the digital image of the 6-well
plate on Adobe Photoshop at 300% view.
FIG. 11 shows growth curve inVero cells of rDEN4 viruses encoding
single Vero adaptation mutations. Vero cells were infected with the
indicated viruses at an MOI of 0.01. Confluent cell monolayers in
25-cm.sup.2 tissue culture flasks were washed and overlaid with a
1.5 ml inoculum containing the indicated virus. After a two hour
incubation at 37.degree. C., cells were washed three times in PBS
and 5 ml of culture medium supplemented with 2% FBS was added. A 1
ml aliquot of tissue culture medium was removed, replaced with
fresh medium, and designated the 0 hour time-point. At the
indicated time points post-infection, samples of tissue culture
medium were removed, clarified, and frozen at -70.degree. C. The
level of virus replication was assayed by plaque titration in Vero
cells. Briefly, serial ten-fold dilutions of cell culture media
samples were inoculated onto confluent Vero cell monolayers in
24-well plates in duplicate and overlaid with Opti-MEM containing
0.8% methylcellulose. After five days, plaques were visualized by
immunoperoxidase staining as described in Example 1. Limit of
detection (L.O.D.) is .gtoreq.0.7 log.sub.10PFU/ml.
FIG. 12 shows growth curve in Vero cells of rDEN4 viruses encoding
combined Vero cell adaptation mutations. Vero cells were infected
with the indicated viruses at an MOI of 0.01. Confluent cell
monolayers in 25-cm.sup.2 tissue culture flasks were washed and
overlaid with a 1.5 ml inoculum containing the indicated virus.
After a two hour incubation at 37.degree. C., cells were washed
three times in PBS and 5 ml of culture medium supplemented with 2%
FBS was added. A 1 ml aliquot of tissue culture medium was removed,
replaced with fresh medium, and designated the 0 hour time-point.
At the indicated time points post-infection, samples of tissue
culture medium were removed, clarified, and frozen at -70.degree.
C. The level of virus replication was assayed by plaque titration
in Vero cells. Limit of detection (L.O.D.) is .gtoreq.0.7
log.sub.10PFU/ml.
BRIEF DESCRIPTION OF THE TABLES
Table 1. Susceptibility of mice to intracerebral DEN4 infection is
age-dependent.
Table 2. Temperature-sensitive (ts) and mouse brain attenuation
(att) phenotypes of 5-FU mutant DEN4 viruses.
Table 3. Nucleotide and amino acid differences of the 5-FU mutant
viruses which are ts in both Vero and HuH-7 cells.
Table 4. Nucleotide and amino acid differences of the 5-FU mutant
viruses which are ts in only HuH-7 cells.
Table 5. Mutations which are represented in multiple 5-FU mutant
DEN4 viruses.
Table 6. Addition of ts mutation 4995 to rDEN4.DELTA.30 confers a
ts phenotype and further attenuates its replication in suckling
mouse brain.
Table 7. Temperature-sensitive (ts) and mouse brain attenuation
(att) phenotypes of 5-FU DEN4 mutant viruses which exhibit a small
plaque (sp) phenotype.
Table 8. Viruses with both ts and sp phenotypes are more restricted
in replication in mouse brain than those with only a ts
phenotype.
Table 9. Nucleotide and amino acid differences of the 5-FU mutant
DEN4 viruses which produce small plaques in both Vero and HuH-7
cells.
Table 10. Nucleotide and amino acid differences of the 5-FU mutant
DEN4 viruses which produce small plaques in only HuH-7 cells.
Table 11. Putative Vero cell adaptation mutations derived from the
full set of 5-FU mutant viruses.
Table 12. Mutagenic oligonucleotides used to generate recombinant
DEN4 viruses containing single 5-FU mutations.
Table 13. sp, ts and mouse attenuation phenotypes of rDEN4 mutant
viruses encoding single mutations identified in six sp 5-FU mutant
viruses.
Table 14. Phenotypes of rDEN4 mutant viruses encoding single
mutations identified in 10 5-FU mutant viruses that are ts in both
Vero and HuH-7 cells.
Table 15. sp, ts and mouse attenuation phenotypes of rDEN4 mutant
viruses encoding single mutations identified in 3 HuH-7
cell-specific ts 5-FU mutant viruses.
Table 16. Temperature-sensitive (ts) and mouse brain attenuation
(att) phenotypes of additional rDEN4 viruses encoding single 5-FU
mutations.
Table 17. Growth of wt DEN-4 2A-13 in SCID mice transplanted with
HuH-7 cells.
Table 18. Combination of ts mutations, NS3 4995 and NS5 7849, in
rDEN4 results in an additive ts phenotype.
Table 19. The 5-FU mutations are compatible with the .DELTA.30
mutation for replication in the brain of suckling mice.
Table 20. Temperature-sensitive and mouse brain attenuation
phenotypes of viruses bearing charge-cluster-to-alanine mutations
in the NS5 gene of DEN4.
Table 21. SCID-HuH-7 attenuation phenotypes of viruses bearing
charge-cluster-to-alanine mutations in the NS5 gene of DEN4.
Table 22. Combination of paired charge-cluster-to-alanine mutations
into double-pair mutant viruses.
Table 23. Temperature-sensitive and mouse brain attenuation
phenotypes of double charge-cluster-to-alanine mutants of the NS5
gene of rDEN4.
Table 24. SCID-HuH-7 attenuation phenotypes of double
charge-cluster-to-alanine mutants of the NS5 gene of rDEN4.
Table 25. Phenotypes (temperature sensitivity, plaque size and
replication in mouse brain and SCID-HuH-7 mice) of wt DEN4 and
viruses containing the .DELTA.30 and 7129 mutations.
Table 26. The 5-fluorouracil 5-1A1 small plaque mutant demonstrates
a restriction of midgut infection following oral infection of Aedes
aegypti mosquitoes.
Table 27. The 5-fluorouracil 5-1A1 small plaque mutant demonstrates
a restriction of infection following intrathoracic inoculation of
Toxorhynchites splendens mosquitoes.
Table 28. Mutagenesis primers for the deletion or swap of sequences
in DEN4 showing conserved differences from tick-borne
flaviviruses.
Table 29. Virus titer and plaque size of 3' UTR mutant viruses in
Vero and C6/36 cells.
Table 30. Infectivity of wt DEN4 and 3' UTR mutants for
Toxorhynchites splendens via intrathoracic inoculation.
Table 31. Infectivity of 3' UTR swap mutant viruses for Aedes
aegypti fed on an infectious bloodmeal.
Table 32. Putative Vero cell adaptation mutations derived from the
set of 5-FU mutant viruses and other DEN4 viruses passaged in Vero
cells.
Table 33. Sequence analysis of rDEN2/4.DELTA.30 clone
27(p4)-2-2A2.
Table 34. Sequence analysis of rDEN2/4.DELTA.30 clone
27(p3)-2-1A1.
Table 35. Recombinant virus rDEN2/4.DELTA.30 bearing Vero
adaptation mutations can be recovery and titered on Vero cells.
Table 36. Putative Vero cell adaptation mutations of dengue type 4
virus and the corresponding wildtype amino acid residue in other
dengue viruses.
Table 37. Mutations known to attenuate dengue type 4 virus and the
corresponding wildtype amino acid residue in other dengue
virus.
BRIEF DESCRIPTION OF THE APPENDICES
Appendix 1. Sequence of recombinant dengue type 4 virus strain 2A
(amino acid sequence SEQ ID NO: 13 and nucleotide sequence SEQ ID
NO: 14).
Appendix 2. Sequence of recombinant dengue type 4 virus strain
rDEN4 (amino acid sequence SEQ ID NO: 15 and nucleotide sequence
SEQ ID NO: 16).
Appendix 3. Sequence of recombinant dengue type 2 chimeric virus
strain rDEN2/4.DELTA.30 (amino acid sequence SEQ ID NO: 17 and
nucleotide sequence SEQ ID NO: 18).
Appendix 4. Alignment of dengue virus polyproteins. DEN4 (SEQ ID
NO: 19); DEN1-WP (SEQ ID NO: 20); DEN2-NGC (SEQ ID NO: 21);
DEN3-H87 (SEQ ID NO: 22).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
To assemble a collection of useful mutations for incorporation in
recombinant live dengue virus vaccines, site-directed and random
mutagenesis techniques were used to introduce mutations into the
dengue virus genome. The resulting mutant viruses were screened for
several valuable phenotypes, including temperature sensitivity in
Vero cells or human liver cells, host cell restriction in mosquito
cells or human liver cells, host-cell adaptation for improved
replication in Vero cells, and attenuation in mice. The genetic
basis for each observed phenotype was determined by direct sequence
analysis of the virus genome. Mutations identified through these
sequencing efforts have been further evaluated by their
re-introduction, singly, or in combination, into recombinant dengue
virus and characterization of the resulting phenotypes. In this
manner, a menu of mutations was developed that is useful in
fine-tuning the attenuation and growth characteristics of dengue
virus vaccines.
Example 1
Chemical Mutagenesis of Dengue Virus Type 4 Yields
Temperature-Sensitive and Attenuated Mutant Viruses
A recombinant live attenuated dengue virus type 4 (DEN4) vaccine
candidate, 2A.DELTA.30, was found previously to be generally
well-tolerated in humans, but a rash and an elevation of liver
enzymes in the serum occurred in some vaccines. 2A.DELTA.30, a
non-temperature-sensitive (ts) virus, contains a 30 nucleotide
deletion in the 3' untranslated region (UTR) of the viral genome.
In the present study, chemical mutagenesis of DEN4 has been
utilized to generate attenuating mutations which may be useful to
further attenuate the incompletely attenuated 2A.DELTA.30 candidate
vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the
presence of 5-fluorouracil, and, from a panel of 1,248 clones that
were isolated in Vero cells, twenty ts mutant viruses were
identified which were ts in both Vero and HuH-7 cells (n=13) or in
HuH-7 cells only (n=7). Each of the twenty ts mutations possessed
an attenuation (att) phenotype as indicated by restricted
replication in the brains of seven day old mice. The complete
nucleotide sequence of the 20 ts mutant viruses identified
nucleotide substitutions in structural and non-structural genes as
well as in the 5' and 3' UTR with more than one change occurring,
in general, per mutant virus. A ts mutation in the NS3 protein
(nucleotide position 4,995) was introduced into a recombinant DEN4
virus possessing the .DELTA.30 deletion creating the
rDEN4.DELTA.30-4995 recombinant virus which was found to be ts and
to be more attenuated than rDEN4.DELTA.30 in the brains of mice. A
menu of attenuating mutations is being assembled that should be
useful in generating satisfactorily attenuated recombinant dengue
vaccine viruses and in increasing our understanding of the
pathogenesis of dengue virus.
The mosquito-borne dengue (DEN) viruses (serotypes 1 to 4) are
members of the Flavivirus genus and contain a single-stranded
positive-sense RNA genome of approximately 10,600 nucleotides (nt)
(Monath, T. P. & Heinz, F. X. 1996 in: Fields Virology B. N.
Fields, et al. Eds. pp. 961-1034 Lippincott-Ravan Publishers,
Philadelphia). The genome organization of DEN viruses is
5'-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3'
(UTR--untranslated region, C--capsid, PrM--pre-membrane,
E--envelope, NS--non-structural) (Chang, G.-J. 1997 in: Dengue and
dengue hemorrhagic fever D. J. Gubler & G. Kuno, eds. pp.
175-198 CAB International, New York; Rice, C. M. 1996 in: Fields
Virology B. N. Fields et al. Eds. pp. 931-959 Lippincott-Raven
Publishers, Philadelphia). A single viral polypeptide is
co-translationally processed by viral and cellular proteases
generating three structural proteins (C, M, and E) and seven NS
proteins. The disease burden associated with DEN virus infection
has increased over the past several decades in tropical and
semitropical countries. Annually, there are an estimated 50-100
million cases of dengue fever (DF) and 500,000 cases of the more
severe and potentially lethal dengue hemorrhagic fever/dengue shock
syndrome (DHF/DSS) (Gubler, D. J. & Meltzer, M. 1999 Adv Virus
Res 53:35-70).
The site of viral replication in DEN virus-infected humans and the
pathogenesis of DF and DHF/DSS are still incompletely understood
(Innis, B. L. 1995 in: Exotic viral infections J. S. Porterfield,
ed. pp. 103-146 Chapman and Hall, London). In humans, DEN virus
infects lymphocytes (Kurane, I. et al. 1990 Arch Virol 110:91-101;
Theofilopoulos, A. N. et al. 1976 J Immunol 117:953-61),
macrophages (Halstead, S. B. et al. 1977 J Exp Med 146:218-29;
Scott, R. M. et al. 1980 J Infect Dis 141:1-6), dendritic cells
(Libraty, D. H. et al. 2001 J Virol 75:3501-8; Wu, S. J. et al.
2000 Nat Med 6:816-20), and hepatocytes (Lin, Y. L. et al. 2000 J
Med Virol 60:425-31; Marianneau, P. et al. 1996 J Gen Virol
77:2547-54). The liver is clearly involved in DEN virus infection
of humans, as indicated by the occurrence of transient elevations
in serum alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) levels in the majority of dengue
virus-infected patients and by the presence of hepatomegaly in some
patients (Kalayanarooj, S. et al. 1997 J Infect Dis 176: 313-21;
Kuo, C. H. et al. 1992 Am J Trop Med Hyg 47:265-70; Mohan, B. et
al. 2000 Trop Pediatr 46:40-3; Wahid, S. F. et al. 2000 Southeast
Asian J Trop Med Public Health 31:259-63). DEN virus
antigen-positive hepatocytes are seen surrounding areas of necrosis
in the liver of fatal cases (Couvelard, A. et al. 1999 Hum Pathol
30:1106-10; Huerre, M. R. et al. 2001 Virchows Arch 438:107-15),
and dengue virus sequences were identified in such cases using
RT-PCR (Rosen, L. et al. 1999 Am J Trop Med Hyg 61:720-4). Of
potential importance to the etiology of severe dengue virus
infection, three studies have demonstrated that the mean levels of
serum ALT/AST were significantly increased in patients with DHF/DSS
versus those with DF (Kalayanarooj, S. et al. 1997 J Infect Dis
176:313-21; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S.
F. et al. 2000 Southeast Asian J Trop Med Public Health
31:259-63).
A vaccine for DEN viruses is not presently licensed. Since previous
infection with one dengue virus serotype can increase the risk for
DHF/DSS following infection with a different serotype (Burke, D. S.
et al. 1988 Am J Trop Med Hyg 38:172-80; Halstead, S. B. et al.
1969 Am J Trop Med Hyg 18:997-1021; Thein, S. et al. 1997 Am J Trop
Med Hyg 56:566-72), it is clear that a dengue virus vaccine will
need to protect against each of the four dengue virus serotypes,
namely DEN1, DEN2, DEN3, and DEN4. Several strategies are currently
being actively pursued in the development of a live attenuated
tetravalent DEN virus vaccine (Bancroft, W. H. et al. 1984 J Infect
Dis 149:1005-10; Bhamarapravati, N. & Sutee, Y. 2000 Vaccine
18:44-7; Guirakhoo, F. et al. 2000 J Virol 74:5477-85; Huang, C. Y.
et al. 2000 J Virol 74:3020-8). Recently, we demonstrated that a
live attenuated DEN4 vaccine candidate, 2A.DELTA.30, was attenuated
and immunogenic in a group of 20 human volunteers (see Example 8).
This recombinant DEN4 virus contains a 30 nt deletion in the 3' UTR
which removes nucleotides 10,478-10,507 and was restricted in
replication in rhesus monkeys. Levels of viremia in humans were low
or undetectable, and virus recovered from the vaccinees retained
the .DELTA.30 mutation. An asymptomatic rash was reported in 50% of
patients. The only laboratory abnormality observed was an
asymptomatic, transient rise in the serum ALT level in 5 of 20
vaccinees. All vaccinees developed serum-neutralizing antibody
against DEN4 virus (mean titer: 1:580). Importantly, 2A.DELTA.30
was not transmitted to mosquitoes fed on vaccinees and has
restricted growth properties in mosquitoes (Troyer, J. M. et al.
2001 Am J Trop Med Hyg 65:414-9). The presence of a rash and of the
elevated ALT levels suggests that the 2A.DELTA.30 vaccine candidate
is slightly under-attenuated in humans. Because of the overall set
of desirable properties conferred by the .DELTA.30 mutation,
chimeric vaccine candidates are being constructed which contain the
structural genes of dengue virus type 1, 2, and 3 and the DEN4
attenuated backbone bearing the genetically stable .DELTA.30
mutation.
Although the initial findings indicate the utility of the
2A.DELTA.30 vaccine candidate, many previous attempts to develop
live attenuated dengue virus vaccines have yielded vaccine
candidates that were either over- or under-attenuated in humans
(Eckels, K. H. et al. 1984 Am J Trop Med Hyg 33:684-9;
Bhamarapravati, N. & Yoksan, S. 1997 in: Dengue and dengue
hemorrhagic fever D. J. Gubler & G. Kuno eds. pp. 367-377 CAB
International, New York; Innis, B. L. et al. 1988 J Infect Dis
158:876-80; McKee, K. T., Jr. et al. 1987 Am J Trop Med Hyg
36:435-42). Therefore, we developed a menu of point mutations which
confer temperature-sensitive (ts) and attenuation (att) phenotypes
upon DEN4. These mutations are envisioned as being useful to
attenuate DEN4 viruses to different degrees and therefore as having
purpose in fine-tuning the level of attenuation of vaccine
candidates such as 2A.DELTA.30. Addition of such mutations to
2A.DELTA.30 or to other dengue virus vaccine candidates is
envisioned as resulting in the generation of a vaccine candidate
that exhibits a satisfactory balance between attenuation and
immunogenicity for humans.
In the present example, chemical mutagenesis of DEN4 has been
utilized to identify point mutations which confer the ts phenotype,
since such viruses often are attenuated in humans. Additionally,
because of the reported involvement of the liver in natural dengue
infection and the elevated ALT levels in a subset of 2A.DELTA.30
vaccinees, mutagenized DEN4 viruses were also evaluated for ts
phenotype in HuH-7 liver cells derived from a human hepatoma. Here,
we describe the identification of 20 DEN4 ts mutant viruses each of
which replicates efficiently in Vero cells, the proposed substrate
for vaccine manufacture, and each of which is attenuated in mice.
Finally, the feasibility of modifying the attenuation phenotype of
the 2A.DELTA.30 vaccine candidate by introduction of a point
mutation in NS3 is demonstrated.
Cells and viruses. WHO Vero cells (African green monkey kidney
cells) were maintained in MEM (Life Technologies, Grand Island,
N.Y.) supplemented with 10% fetal bovine serum (FBS) (Summit
Biotechnologies, Fort Collins, Colo.), 2 mM L-glutamine (Life
Technologies), and 0.05 mg/ml gentamicin (Life Technologies). HuH-7
cells (human hepatoma cells) (Nakabayashi, H. et al. 1982 Cancer
Res 42:3858-63) were maintained in D-MEM/F-12 (Life Technologies)
supplemented with 10% FBS, 1 mM L-glutamine and 0.05 mg/ml
gentamicin. C6/36 cells (Aedes albopictus mosquito cells) were
maintained in complete MEM as described above supplemented with 2
mM non-essential amino acids (Life Technologies).
The wild type (wt) DEN4 2A virus was derived from a cDNA clone of
DEN4 strain 814669 (Dominica, 1981) (Men, R. et al. 1996 J Virol
70:3930-7). Sequence of the cDNA of DEN 4 2A virus is presented in
Appendix 1. The full-length 2A cDNA clone has undergone several
subsequent modifications to improve its ability to be genetically
manipulated. As previously described, a translationally-silent XhoI
restriction enzyme site was engineered near the end of the E region
at nucleotide 2348 to create clone 2A-XhoI (Bray, M. & Lai, C.
J. 1991 PNAS USA 88:10342-6). The viral coding sequence of the
2A-XhoI cDNA clone was further modified using site-directed
mutagenesis to create clone p4: a unique BbvCI restriction site was
introduced near the C-prM junction (nucleotides 447-452); an extra
XbaI restriction site was ablated by mutation of nucleotide 7730;
and a unique SacII restriction site was created in the NS5 region
(nucleotides 9318-9320). Each of these engineered mutations is
translationally silent and does not change the amino acid sequence
of the viral polypeptide. Also, several mutations were made in the
vector region of clone p4 to introduce or ablate additional
restriction sites. The cDNA clone p4.DELTA.30 was generated by
introducing the .DELTA.30 mutation into clone p4. This was
accomplished by replacing the MluI-KpnI fragment of p4 (nucleotides
10403-10654) with that derived from plasmid 2A.DELTA.30 containing
the 30 nucleotide deletion. The cDNA clones p4 and p4.DELTA.30 were
subsequently used to generate recombinant viruses rDEN4 (Appendix
2) and rDEN4.DELTA.30, respectively. (The GenBank accession number
for rDEN4 is AF326825 and the accession for rDEN4.DELTA.30 is
AF326827).
Chemical mutagenesis of DEN4. Confluent monolayers of Vero cells
were infected with wt DEN4 2A at an multiplicity of infection (MOI)
of 0.01 and incubated for 2 hours at 32.degree. C. Infected cells
were then overlaid with MEM supplemented with 2% FBS and
5-fluorouracil (5-FU) (Sigma, St. Louis, Mo.) at concentrations
ranging from 10 mM to 10 nM. After incubation at 32.degree. C. for
five days, cell culture medium was harvested, clarified by
centrifugation, and frozen at -70.degree. C. Clarified supernatants
were then assayed for virus titer by plaque titration in Vero
cells. Serial ten-fold dilutions of the clarified supernatant were
prepared in Opti-MEM I (Life Technologies) and inoculated onto
confluent Vero cell monolayers in 24-well plates. After incubation
at 35.degree. C. for two hours, monolayers were overlaid with 0.8%
methylcellulose (EM Science, Gibbstown, N.J.) in Opti-MEM I
supplemented with 2% FBS, gentamicin, and L-glutamine. Following
incubation at 35.degree. C. for five days, plaques were visualized
by immunoperoxidase staining. Vero cell monolayers were fixed in
80% methanol for 30 minutes and washed for 10 minutes with antibody
buffer which consists of 3.5% (w/v) nonfat dry milk (Nestle, Solon,
Ohio) in phosphate buffered saline (PBS). Cells were then incubated
for one hour at 37.degree. C. with an anti-DEN4 rabbit polyclonal
antibody preparation (PRNT.sub.50 of >1:2000) diluted 1:1,000 in
antibody buffer. After one wash with antibody buffer, cells were
incubated for one hour with peroxidase-labeled goat-anti-rabbit IgG
(KPL, Gaithersburg, Md.) diluted 1:500 in antibody buffer.
Monolayers were washed with PBS, allowed to dry briefly, overlaid
with peroxidase substrate (KPL), and plaques were counted.
Virus yields in cultures treated with 1 mM 5-FU were reduced
100-fold compared to untreated cultures, and the virus present in
the supernatant from the 1 mM 5-FU-treated culture was terminally
diluted to derive clones for phenotypic characterization. Briefly,
96 well plates of Vero cells were inoculated with the 5-FU-treated
virus at an MOI that yielded 10 or fewer virus-positive wells per
plate. After a five-day incubation at 35.degree. C., cell culture
media from the 96 well plates were temporarily transferred to 96
well plates lacking cells, and the positive cultures were
identified by immunoperoxidase staining of the infected-cell
monolayers. Virus from each positive well was transferred to
confluent Vero cell monolayers in 12 well plates for amplification.
Cell culture medium was harvested from individual wells five or six
days later, clarified by centrifugation, aliquoted to 96 deep-well
polypropylene plates (Beckman, Fullerton, Calif.) and frozen at
-70.degree. C. A total of 1,248 virus clones were prepared from the
1 mM 5-FU-treated cultures. Two wt virus clones, 2A-1 and 2A-13,
were generated in the same manner from the 5-FU untreated control
cultures.
Screening of clones for ts and att phenotypes. The 1,248 virus
clones were screened for ts phenotype by assessing virus
replication at 35.degree. C. and 39.degree. C. in Vero and HuH-7
cells. Cell monolayers in 96 well plates were inoculated with
serial ten-fold dilutions of virus in L-15 media (Quality
Biologicals, Gaithersburg, Md.) supplemented with 2% FBS,
L-glutamine and gentamicin. Cells were incubated at the indicated
temperatures for five days in temperature-controlled water baths,
and presence of virus was determined by immunoperoxidase staining
as described above. Virus clones which demonstrated a 100-fold or
greater reduction in titer at 39.degree. C. were terminally diluted
an additional two times and amplified in Vero cells. The efficiency
of plaque formation (EOP) at permissive and restrictive
temperatures of each triply biologically cloned virus suspension
was determined as follows. Plaque titration in Vero and HuH-7 cells
was performed as described above except virus-infected monolayers
were overlaid with 0.8% methylcellulose in L-15 medium supplemented
with 5% FBS, gentamicin, and L-glutamine. After incubation of
replicate plates for five days at 35, 37, 38, or 39.degree. C. in
temperature-controlled water baths, plaques were visualized by
immunoperoxidase staining and counted.
The replication of DEN4 5-FU ts mutant viruses was evaluated in
Swiss Webster suckling mice (Taconic Farms, Germantown, N.Y.).
Groups of six one-week-old mice were inoculated intracranially with
10.sup.4 PFU of virus diluted in 30 .mu.l Opti-MEM I. Five days
later, mice were sacrificed and brains were removed and
individually homogenized in a 10% suspension of phosphate-buffered
Hank's balanced salt solution containing 7.5% sucrose, 5 mM sodium
glutamate, 0.05 mg/ml ciprofloxacin, 0.06 mg/ml clindamycin, and
0.0025 mg/ml amphotericin B. Clarified supernatants were frozen at
-70.degree. C. and subsequently virus titer was determined by
titration in Vero cells, and plaques were stained by the
immunoperoxidase method described above.
Sequence analysis of viral genomes. The nucleotide sequence of the
5-FU-mutagenized DEN4 viruses was determined. Briefly, genomic
viral RNA was isolated from virus clones with the QIAamp viral RNA
mini kit (Qiagen, Valencia, Calif.) and reverse transcription was
performed using the SuperScript First Strand Synthesis System for
RT-PCR (Life Technologies) and random hexamer primers. Advantage
cDNA polymerase (Clontech, Palo Alto, Calif.) was used to generate
overlapping PCR fragments of approximately 2,000 nt which were
purified by HighPure PCR Product Purification System (Roche
Diagnostics, Indianapolis, Ind.). DEN-specific primers were used in
BigDye terminator cycle sequencing reactions (Applied Biosystems,
Foster City, Calif.) and reactions were analyzed on a 3100 genetic
analyzer (Applied Biosystems). Primers were designed to sequence
both strands of the PCR product from which consensus sequences were
assembled.
The nucleotide sequence of the 5' and 3' regions of the viral
genome were determined as above after circularization of the RNA
genome. The 5' cap nucleoside of the viral RNA was excised using
tobacco acid pyrophosphatase (Epicentre Technologies, Madison,
Wis.) and the genome was circularized by RNA ligase (Epicentre
Technologies). A RT-PCR fragment was generated which overlapped the
ligation junction (5' and 3' ends) and was sequenced as described
above.
Generation of recombinant DEN4 viruses. The mutation at nt position
4,995 in NS3 was introduced into the p4 cDNA construct by
site-directed mutagenesis (Kunkel, T. A. 1985 PNAS USA 82:488-92).
The StuI-BstBI (nt 3,619-5,072) fragment of p4 was sub-cloned into
a modified pUC119 vector. The U>C mutation at nt position 4,995
was engineered by site-directed mutagenesis into the p4 fragment,
cloned back into the p4 cDNA construct, and the presence of the
mutation was confirmed by sequence analysis. The .DELTA.30 mutation
was introduced into the 3' UTR of the p4-4995 cDNA clone by
replacing the MluI-KpnI fragment with that derived from the
p4.DELTA.30 cDNA clone, and the presence of the deletion was
confirmed by sequence analysis. Full length RNA transcripts were
prepared from the above cDNA clones by in vitro transcription.
Briefly, transcription consisted of a 50 .mu.l reaction mixture
containing 1 .mu.g linearized plasmid, 60 U SP6 polymerase (New
England Biolabs (NEB), Beverly, Mass.), 1.times.RNA polymerase
buffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl.sub.2, 2 mM spermidine,
10 mM dithiothreitol), 0.5 mM m7G(5')ppp(5')G cap analog (NEB), 1
mM each nucleotide triphosphate, 1 U pyrophosphatase (NEB), and 80
U RNAse inhibitor (Roche, Indianapolis, Ind.). This reaction
mixture was incubated at 40.degree. C. for 90 min and the resulting
transcripts were purified using RNeasy mini kit (Qiagen, Valencia,
Calif.).
For transfection of C6/36 cells, RNA transcripts were combined with
DOTAP liposomal transfection reagent (Roche) in HEPES-buffered
saline (pH 7.6) and added to cell monolayers in 6 well plates.
After incubation at 32.degree. C. for 12-18 hours, the cell culture
media were removed and replaced with MEM supplemented with 5% FBS,
L-glutamine, gentamicin and non-essential amino acids. Cell
monolayers were incubated for an additional 5 to 7 days and cell
culture media were harvested, clarified by centrifugation, and
assayed for the presence of virus by plaque titration in Vero
cells. Recovered viruses were terminally diluted twice as described
above, and virus suspensions for further analysis were prepared in
Vero cells.
In vitro (tissue culture) and in vivo replication of wt DEN4 and
DEN4.DELTA.30. The level of replication of both wt DEN4 2A and the
vaccine candidate, 2A.DELTA.30, was evaluated in Vero (monkey
kidney) and HuH-7 (human hepatoma) cells (FIG. 1), the latter of
which has recently been found to efficiently support the
replication of DEN2 virus (Lin, Y. L. et al. 2000 J Med Virol
60:425-31). The pattern of replication of wt DEN4 2A and
2A.DELTA.30 was similar in both cell lines. Viral titers from
cultures infected with 2A.DELTA.30 at an MOI of 0.01 were slightly
reduced compared to wt DEN4 2A at 72 hours, but at later time
points their level of replication was equivalent. The efficient
replication of both DEN4 viruses in each cell line indicated that
these continuous lines of cells would be useful for
characterization of the ts phenotype of the 1248 potential mutant
viruses.
The level of replication of DEN4 virus administered intracerebrally
to Swiss Webster mice was first determined to assess whether mice
could be used to efficiently evaluate and quantitate the
attenuation phenotype of a large set of mutant viruses. Since the
susceptibility of mice to DEN infection is age dependent (Cole, G.
A. & Wisseman, C. L. Jr. 1969 Am J Epidemiol 89:669-80; Cole,
G. A. et al. 1973 J Comp Pathol 83:243-52), mice aged 7 to 21 days
were infected with 2A-13 (a clone of DEN4 wild type virus--see
below), rDEN4 or rDEN4.DELTA.30, and after five days the brain of
each mouse was removed, and the level of viral replication was
quantitated by plaque assay (Table 1). The results indicated that
the two wt DEN4 viruses and the rDEN4.DELTA.30 vaccine candidate
replicated to high titer (>6.0 log.sub.10PFU/g brain) in 7-day
old mice and that the mean viral titers were similar among the
three viruses. These results demonstrated the feasibility of using
7-day old mice to screen a large set of mutant viruses, and the
high level of replication of wild type and vaccine candidate
permits one to quantitate the magnitude of the restriction of
replication specified by an attenuating mutation over a 10.000-fold
range.
Generation and in vitro characterization of DEN4 5-FU mutant
viruses. A panel of 1,248 DEN4 virus clones was generated from a
5-FU-mutagenized suspension of wt DEN4 2A as described above (FIG.
2). Each clone was tested in Vero and HuH-7 cells for the ts
phenotype at 39.degree. C., and putative ts mutant viruses were
subjected to two additional rounds of biological cloning by
terminal dilution, and the ts phenotype of each further cloned
virus population was examined in more detail by determining their
efficiency of plating (EOP) at permissive temperature (35.degree.
C.) and at various restrictive temperatures (Table 2). One virus
(clone 2A-13) without a ts phenotype, which was passaged in an
identical fashion as the ts mutant viruses, served as the virus to
which each of the ts mutant viruses was directly compared for both
the ts and att phenotypes.
Thirteen 5-FU mutant viruses were identified which have a ts
phenotype in both Vero and HuH-7 cells, and seven mutant viruses
were ts only in HuH-7 cells (Table 2). Mutant viruses which were ts
in Vero cells but not in HuH-7 cells were not identified.
Temperature-sensitivity was defined as a .gtoreq.2.5 or .gtoreq.3.5
log.sub.10PFU/ml reduction in virus titer in Vero or HuH-7 cells,
respectively, at an indicated temperature when compared to the
permissive temperature of 35.degree. C. Wild type DEN4 2A was found
to have approximately a 0.5 and 1.5 log.sub.10PFU/ml reduction in
virus titer in Vero or HuH-7 cells at 39.degree. C., respectively.
The .DELTA.30 deletion did not confer a ts phenotype in Vero or
HuH-7 cells and exhibited only a slight reduction in virus titer
(2.2 log.sub.10PFU/ml) at 39.degree. C. in HuH-7 cells, which was
less than 10-fold greater than the reduction of wt DEN4 2A at that
temperature. Several 5-FU mutant viruses had a greater than
10.000-fold reduction in virus titer at 39.degree. C. in both Vero
and HuH-7 cells. A complete shut-off in viral replication at
39.degree. C. in HuH-7 cells was observed in five virus clones
(#571, 605, 631, 967, and 992) which were not ts in Vero cells.
Mutations that selectively restrict replication in HuH-7 liver
cells may be particularly useful in controlling the replication of
dengue virus vaccine candidates in the liver of vaccinees.
Replication of DEN4 5-FU mutant viruses in suckling mice. The level
of replication of each of the 20 ts DEN4 mutant viruses in mouse
brain was determined (Table 2). The titers obtained were compared
to that of the two wt viruses, 2A-13 and rDEN4, which each
replicated to a level of greater than 10.sup.6 PFU/g of brain
tissue, and to that of the 2A.DELTA.30 mutant, which conferred only
a limited 0.5 log.sub.10PFU/g reduction in mean virus titer
compared to the wt controls. The observed reduction in the level of
rDEN4.DELTA.30 replication was consistent among 11 separate
experiments. Interestingly, the rDEN4.DELTA.30 virus, which was
attenuated in both rhesus monkeys and humans (Example 8), was only
slightly restricted in replication in mouse brain. Varying levels
of restriction of replication were observed among the mutant
viruses ranging from a 10-fold (#473) to over 6.000-fold (#686)
reduction. Mutant viruses with ts phenotypes in both Vero and HuH-7
cells, as well as in HuH-7 cells alone, were found to have
significant att phenotypes. Five of 13 5-FU mutant viruses with ts
phenotypes in both Vero and HuH-7 cells and five of seven mutant
viruses with ts phenotypes in HuH-7 cells alone had greater than a
100-fold reduction in virus replication. There appeared to be no
direct correlation between the magnitude of the reduction in
replication at restrictive temperature in tissue culture and the
level of attenuation in vivo. The similar level of temperature
sensitivity and replication of the rDEN4 wt and clone 2A-13 in
mouse brain indicated that observed differences in replication
between the ts mutant viruses and clone 2A-13 was not simply a
function of passage in Vero cells, but reflects the sequence
differences between these viruses.
Sequence analysis of DEN4 5-FU mutant viruses. To determine the
genetic basis of the observed ts and att phenotypes, the complete
nucleotide sequence of each ts mutant and of clone 2A-13 was
determined and summarized in Table 3 (ts in Vero and HuH-7 cells)
and Table 4 (ts in only HuH-7 cells).
The only type of mutation identified in the 20 mutant viruses
sequenced was a nucleotide substitution (no deletions or insertions
occurred), and these were present in each of the coding regions
except C and NS4A. Three mutant viruses (#239, 489, and 773)
contained only a single missense point mutation in NS3 at nt
position 4,995 resulting in a Ser to Pro amino acid (a.a.) change
at a.a. position 1,632. For #773, this was the sole mutation
present (Table 3). The non-coding mutations in coding regions are
not considered to be significant. The 17 additional mutant viruses
had multiple mutations (two to five) in a coding region or in an
UTR which could potentially confer the observed ts or att
phenotypes. Five of the 17 mutant viruses with multiple mutations
(#473, 718, 759, 816, and 938) also encoded the point mutation at
nt position 4,995. The presence of the 4,995 mutation was found in
only DEN4 mutant viruses with ts phenotypes in both Vero and HuH-7
cells.
The sequence analysis indicated that 10 mutant viruses which were
ts in Vero and HuH-7 cells and three mutant viruses which were ts
in only HuH-7 cells contained mutations in only the 5' and 3' UTR
and/or in a nonstructural protein. These mutations are especially
suitable for inclusion in chimeric dengue virus vaccine candidates
in which the structural genes derive from a DEN1, DEN2, or DEN3
serotype and the remaining coding and non-coding regions come from
an attenuated DEN4 vector. Mutations identified in 5-FU DEN4 mutant
viruses which were ts in only HuH-7 cells (Table 4) may potentially
be utilized in vaccine candidates, such as rDEN4.DELTA.30, to
selectively control the replication and pathogenesis of DEN4 in the
liver. These combined results from the sequence analysis of 5-FU
mutant viruses demonstrate the utility of chemical mutagenesis as a
means of introducing attenuating mutations into the dengue virus
genome.
The presence of a point mutation at nt position 4,995 in eight
separate mutant viruses was described above. Five additional point
mutations were also represented in multiple viruses including nt
changes at position 1,455 in E, 7,162, 7,163 and 7,564 in NS4B, and
10,275 in the 3' UTR (Table 5). The significance of the occurrence
of these "sister" mutations in multiple viruses is discussed in
Example 6. Interestingly, the wild-type, parallel-passaged virus,
2A-13, also contained a single mutation at the 7,163 nt position in
NS4B.
Introduction of a ts mutation into rDEN4 and rDEN4.DELTA.30. The
presence of a single nucleotide substitution (U>C mutation at nt
position 4,995 in NS3) in three separate mutant viruses (clones
239, 489, and 773) indicated that this mutation specified the ts
and att phenotypes in each of the three mutant viruses. This
mutation was cloned into cDNA construct of p4 and p4.DELTA.30 and
recombinant viruses were recovered and designated rDEN4-4995 and
rDEN4.DELTA.30-4995, respectively. These recombinant viruses were
tested for ts and att phenotypes as described above (Table 6). As
expected, introduction of mutation 4995 into rDEN4 wt resulted in a
significant ts phenotype at 39.degree. C. in both Vero and HuH-7
cells. rDEN4-4995 grew to nearly wild-type levels at the permissive
temperature, 35.degree. C., in both cell types, but demonstrated a
greater than 10,000-fold reduction at 39.degree. C. (shut-off
temperature) in both Vero and HuH-7 cells. The addition of the 4995
mutation to rDEN4.DELTA.30 yields a recombinant virus,
rDEN4.DELTA.30-4995, that exhibits the same level of temperature
sensitivity as rDEN4-4995 (Table 6).
The rDEN4 viruses encoding the 4995 mutation were next tested for
replication in the brains of suckling mice (Table 6). The 4995
mutation conferred an att phenotype upon both rDEN4 and
rDEN4.DELTA.30. There was an approximately 1,000-fold reduction in
virus replication compared to that of wt virus. The combination of
point mutation 4995 and the .DELTA.30 deletion did not appear to
result in an additive reduction of virus replication. These results
confirmed that the 4995 point mutation indeed specifies the ts and
att phenotypes. Importantly, the utility of modifying tissue
culture and in vivo phenotypes of the rDEN4.DELTA.30 vaccine
candidate by introduction of additional mutations was also
demonstrated.
Discussion. Herein we teach how to prepare a tetravalent,
live-attenuated dengue virus vaccine using rDEN4.DELTA.30 as the
DEN4 component and three antigenic chimeric viruses expressing the
structural proteins (C, prM, and E) of DEN1, DEN2, and DEN3 from
the attenuated rDEN4.DELTA.30 vector (Example 8). DEN4 virus
rDEN4.DELTA.30 containing the .DELTA.30 deletion mutation in the 3'
UTR manifests restricted replication in humans while retaining
immunogenicity. Since rDEN4.DELTA.30 retains a low level of
residual virulence for humans despite this restricted replication,
the present study was initiated to generate additional attenuating
mutations that are envisioned as being useful to further attenuate
rDEN4.DELTA.30 or other dengue viruses and that are envisioned as
being incorporated into any of the three antigenic chimeric viruses
or other dengue viruses as needed. Temperature-sensitive mutants of
dengue viruses (Bhamarapravati, N. & Yoksan, S. 1997 in: Dengue
and Dengue Hemorrhagic Fever D. J. Gubler & G. Kuno eds. pp.
367-377 CAB International, New York; Eckels, K. H. et al. 1980
Infect Immun 27:175-80) as well of other viruses (Skiadopoulos, M.
H. et al. 1998 J Virol 72:1762-8; Whitehead, S. S. et al. 1999 J
Virol 73:871-7) manifest restricted replication in vivo. We have
generated a panel of 20 ts DEN4 mutant viruses, determined their
genomic sequence, and assessed their in vivo attenuation
phenotypes. The 20 ts DEN4 mutant viruses were generated by growth
in the presence of 5-FU and were first selected for viability in
Vero cells, the substrate planned for use in the manufacture of
these vaccines, to ensure that the mutant viruses can be grown
efficiently in a suitable substrate.
Two classes of mutant viruses were obtained; those ts in both Vero
and HuH-7 cells (n=13) or those ts in only HuH-7 cells (n=7). The
viruses exhibited a range in their level of temperature sensitivity
from a 100- to 1,000,000-fold reduction in replication at the
restrictive temperature of 39.degree. C. Since our DEN4 vaccine
candidate retains a low level of virulence for the liver and other
findings support the ability of dengue viruses to infect
hepatocytes (Lin, Y. L. et al. 2000 J Med Virol 60:425-31;
Marianneau, P. et al. 1997 J Virol 71:3244-9) and cause liver
pathology (Couvelard, A. et al. 1999 Hum Pathol 30:1106-10; Huerre,
M. R. et al. 2001 Virchows Arch 438:107-15), we sought to develop
mutations that would selectively restrict replication of dengue 4
virus in liver cells. Toward this end, we identified seven mutant
viruses which have a HuH-7 cell-specific ts phenotype. The
mutations present in these viruses are the first reported in DEN
viruses that confer restricted replication in liver cells and are
envisioned as being useful in limiting virus replication and
pathogenesis in the liver of vaccine recipients. The contribution
of individual mutations identified in the HuH-7 cell-specific ts
viruses to the observed phenotypes is envisioned as being assessed
by introduction of the individual mutations into recombinant DEN4
viruses.
Recent evidence has indicated that the magnitude of the viremia in
DEN-infected patients positively correlates with disease severity,
i.e., the higher the titer of viremia the more severe the disease
(Murgue, B. et al. 2000 J Med Virol 60:432-8; Vaughn, D. W. et al.
2000 J Infect Dis 181:2-9). This indicates that mutations that
significantly restrict replication of vaccine candidates in vivo
are the foundation of a safe and attenuated vaccine. Evaluation of
DEN virus vaccine candidates for in vivo attenuation is complicated
by the lack of a suitable animal model which accurately mimics the
disease caused by dengue viruses in humans. In the absence of such
a model, the replication of the panel of 5-FU mutant viruses in the
brains of Swiss Webster suckling mice was assessed as a means to
identify an in vivo attenuation phenotype since this animal model
is well-suited for the evaluation of a large set of mutant viruses.
Each of the 20 ts mutant viruses exhibited an att phenotype
manifesting a 10- to 6.000-fold reduction in replication in the
brain of mice as compared to wt DEN4 virus (Table 2). This
indicates that there is a correlation between the presence of the
ts phenotype in tissue culture and attenuation of the mutant in
vivo confirming the utility of selecting viruses with this marker
as vaccine candidates. However, there was no correlation between
the level of temperature sensitivity and the level of restriction
in vivo. Furthermore, Sabin observed a dissociation between mouse
neurovirulence and attenuation in humans by generating an effective
live attenuated virus vaccine against DEN by passage of virus in
mouse brain. This research actually resulted in a highly
mouse-neurotropic DEN virus which, paradoxically, was significantly
attenuated in humans (Sabin, A. B. 1952 Am J Trop Med Hyg 1:30-50).
Despite this, attenuation for the suckling mouse brain has been
reported for other live-attenuated DEN virus vaccine candidates
including the DEN2 PDK-53 vaccine strain which is non-lethal in
mice and DEN-2 PR-159/S-1 vaccine strain which was significantly
attenuated compared to its parental wild-type virus
(Bhamarapravati, N. & Yoksan, S. 1997 in: Dengue and Dengue
Hemorrhagic Fever D. J. Gubler & G. Kuno eds. pp. 367-377 CAB
International, New York; Butrapet, S. et al. 2000 J Virol
74:3011-9; Eckels, K. H. et al. 1980 Infect Immun 27:175-80; Innis,
B. L. et al. 1988 J Infect Dis 158:876-80). Replication in rhesus
monkeys has been reported to be predictive of attenuation for
humans (Innis, B. L. et al. 1988 J Infect Dis 158:876-80).
Recently, murine models of DEN virus infection have been developed
using SCID mice transplanted with human macrophage (Lin, Y. L. et
al. 1998 J Virol 72:9729-37) or liver cell lines (An, J. et al.
1999 Virology 263:70-7), but these mice have not as yet been used
to assess att phenotypes of candidate vaccine viruses. Mutant
viruses or recombinant viruses bearing one or more of these
mutations described herein are envisioned as being tested for
replication in rhesus monkeys (or other suitable animal model) as
predictive for attenuation in humans.
The chemical mutagenesis of DEN4 virus and sequence analysis of
resulting viruses described here has resulted in the identification
of a large number of point mutations resulting in amino acid
substitutions in all genes except C and NS4A as well as point
mutations in the 5' and 3' UTR (Tables 3 and 4). This approach of
whole-genome mutagenesis has the benefit of identifying mutations
dispersed throughout the entire genome which are pre-selected for
viability in the Vero cell substrate. Ten 5-FU mutant viruses which
were ts in Vero and HuH-7 cells and three viruses which were
selectively ts in HuH-7 cells contained only mutations outside of
the genes encoding the structural proteins, i.e., in the 5' and 3'
UTR or NS genes. These mutations along with the .DELTA.30 deletion
in the 3' UTR are particularly suited for inclusion in antigenic,
chimeric vaccines which consist of an attenuated DEN4 vector
bearing the wild-type structural genes (C, prM, E) of the other DEN
virus serotypes. Use of this strategy has several advantages. Each
antigenic chimeric virus that possesses structural proteins from a
wild-type virus along with attenuating mutations in their UTRs or
NS genes should maintain its infectivity for humans, which is
mediated largely by the E protein, and, therefore, each vaccine
component should be immunogenic (Huang, C. Y. et al. 2000 J Virol
74:3020-8). The replicative machinery of the tetravalent vaccine
strains would share the same attenuating mutations in the NS genes
or in the UTR which should attenuate each vaccine component to a
similar degree and thereby minimize interference or complementation
among the four vaccine viruses. In addition, wild-type E protein
would be expected to most efficiently induce neutralizing
antibodies against each individual DEN virus.
Sequence analysis of dengue viruses (Blok, J. et al. 1992 Virology
187:573-90; Lee, E. et al. 1997 Virology 232:281-90; Puri, B. et
al. 1997 J Gen Virol 78:2287-91) and yellow fever viruses (Dunster,
L. M. et al. 1999 Virology 261:309-18; Holbrook, M. R. et al. 2000
Virus Res 69:31-9) previously generated by serial passage in tissue
culture have mutations throughout much of the genome, a pattern we
have observed in the present study. Recent analysis of the DEN2
PDK-53 vaccine strain has identified the important mutations
involved in attenuation which were located in non-structural
regions including the 5' UTR, NS1 and NS3 (Butrapet, S. et al. 2000
J Virol 74:3011-9). This DEN2 vaccine strain has been used to
generate a chimeric virus with DEN1 C-prM-E genes (Huang, C. Y. et
al. 2000 J Virol 74:3020-8). In separate studies, the sequence of
the DEN1 vaccine strain 45AZ5 PDK-27 was determined and compared to
parental viruses, but the mutations responsible for attenuation
have not yet been identified (Puri, B. et al. 1997 J Gen Virol
78:2287-91).
Several amino acid substitutions were identified in more than one
ts 5-FU mutant virus (Table 5). Lee et al. have previously reported
finding repeated mutations in separate DEN3 virus clones after
serial passage in Vero cells (Lee, E. et al. 1997 Virology
232:281-90). A mutation (K>N) identified in E at a.a. position
202 in a single DEN3 passage series was also found in our 5-FU
mutant virus #1012 (K>E). Mutations observed in the 5-FU sister
mutant viruses are envisioned as representing adaptive changes that
confer an increased efficiency of DEN4 replication in Vero cells.
Such mutations are envisioned as being beneficial for inclusion in
a live-attenuated DEN virus vaccine by increasing the yield of
vaccine virus during manufacture. Interestingly, three distinct
amino acid substitutions were found in NS4B of the 5-FU sister
mutant viruses. The exact function of this gene is unknown, but
previous studies of live-attenuated yellow fever vaccines
(Jennings, A. D. et al. 1994 J Infect Dis 169:512-8; Wang, E. et
al. 1995 J Gen Virol 76:2749-55) and Japanese encephalitis vaccines
(Ni, H. et al. 1995 J Gen Virol 76:409-13) have identified
mutations in NS4B associated with attenuation phenotypes.
The mutation at nt position 4995 of NS3 (S1632P) was present as the
only significant mutation identified in three 5-FU mutant viruses
(#239, #489, and #773). This mutation was introduced into a
recombinant DEN4 virus and found to confer a ts and att phenotype
(Table 6). These observations clearly identify the 4995 mutation as
an attenuating mutation. Analysis of a sequence alignment (Chang,
G.-J. 1997 in: Dengue and Dengue Hemorrhagic Fever D. J. Gubler
& G. Kuno, eds. pp. 175-198 CAB International, New York) of the
four dengue viruses indicated that the Ser at a.a. position 1632 is
conserved in DEN1 and DEN2, while DEN3 contains an Asn at this
position indicating that the mutation is predicted to be useful in
modifying the phenotypes of the other DEN virus serotypes. The NS3
protein is 618 a.a. in length and contains both serine protease and
helicase activities (Bazan, J. F. & Fletterick, R. J. 1989
Virology 171:637-9; Brinkworth, R. I. et al. 1999 J Gen Virol
80:1167-77; Valle, R. P. & Falgout, B. 1998 J Virol 72:624-32).
The 4995 mutation results in a change at a.a. position 158 in NS3
which is located in the N-terminal region containing the protease
domain. Amino acid position 158 is located two a.a. residues away
from an NS3 conserved region designated homology box four. This
domain has been identified in members of the flavivirus family and
is believed to be a critical determinant of the NS3 protease
substrate specificity (Bazan, J. F. & Fletterick, R. J. 1989
Virology 171:637-9; Brinkworth, R. I. et al. 1999 J Gen Virol
80:1167-77). However, the exact mechanism which results in the
phenotype associated with the 4995 mutation has not yet been
identified. The identification of the 4995 mutation as an
attenuating mutation permits a prediction of its usefulness for the
further attenuation of rDEN4.DELTA.30.
We have determined the contribution of individual 5-FU mutations to
the observed phenotypes by introduction of the mutations into
recombinant DEN4 viruses as was demonstrated herein for the 4995
mutation (see Example 3). In addition, combination of individual
mutations with each other or with the .DELTA.30 mutation is useful
to further modify the attenuation phenotype of DEN4 virus candidate
vaccines. The introduction of the 4995 mutation into rDEN4.DELTA.30
described herein rendered the rDEN4.DELTA.30-4995 double mutant ts
and 1000-fold more attenuated for the mouse brain than
rDEN4.DELTA.30. This observation has demonstrated the feasibility
of modifying both tissue culture and in vivo phenotypes of this and
other dengue virus vaccine candidates. Once the mutations
responsible for the HuH-7 cell-specific ts phenotype are identified
as described above and introduced into the rDEN4.DELTA.30 vaccine
candidate, we envision confirming that these mutations attenuate
rDEN4.DELTA.30 vaccine virus for the liver of humans. A menu of
attenuating mutations is envisioned as being assembled that is
predicted to be useful in generating satisfactorily attenuated
recombinant dengue vaccine viruses and in increasing our
understanding of the pathogenesis of dengue virus (see Example
7).
Example 2
Chemical Mutagenesis of DEN4 Virus Results in Small-Plaque Mutant
Viruses with Temperature-Sensitive and Attenuation Phenotypes
Mutations that restrict replication of dengue virus have been
sought for the generation of recombinant live-attenuated dengue
virus vaccines. Dengue virus type 4 (DEN4) was previously grown in
Vero cells in the presence of 5-fluorouracil, and the
characterization of 1,248 mutagenized, Vero cell-passaged clones
identified 20 temperature-sensitive (ts) mutant viruses that were
attenuated (att) in suckling mouse brain (Example 1). The present
investigation has extended these studies by identifying an
additional 22 DEN4 mutant viruses which have a small-plaque size
(sp) phenotype in Vero cells and/or the liver cell line, HuH-7.
Five mutant viruses have a sp phenotype in both Vero and HuH-7
cells, three of which are also ts. Seventeen mutant viruses have a
sp phenotype in only HuH-7 cells, thirteen of which are also ts.
Each of the sp viruses was growth restricted in the suckling mouse
brain, exhibiting a wide range of reduction in replication (9- to
100,000-fold). Complete nucleotide sequence was determined for the
22 DEN4 sp mutant viruses, and nucleotide substitutions were found
in the 3' untranslated region (UTR) as well as in all coding
regions except NS4A. Identical mutations have been identified in
multiple virus clones indicating that they are involved in the
adaptation of DEN4 virus to efficient growth in Vero cells.
The DEN viruses cause more disease and death of humans than any
other arbovirus, and more than 2.5 billion people live in regions
with endemic dengue infection (Gubler, D. J. 1998 Clin Microbiol
Rev 11:480-96). Annually, there are an estimated 50-100 million
cases of dengue fever (DF) and 500,000 cases of the more severe and
potentially lethal dengue hemorrhagic fever/dengue shock syndrome
(DHF/DSS) (Gubler, D. J. & Meltzer, M. 1999 Adv Virus Res
53:35-70). Dengue fever is an acute infection characterized by
fever, retro-orbital headache, myalgia, and rash. At the time of
defervescence during DF, a more severe complication of DEN virus
infection, DHF/DSS, may occur which is characterized by a second
febrile period, hemorrhagic manifestations, hepatomegaly,
thrombocytopenia, and hemoconcentration, which may lead to
potentially life-threatening shock (Gubler, D. J. 1998 Clin
Microbiol Rev 11:480-96).
The sites of DEN virus replication in humans and their importance
and relationship to the pathogenesis of DF and DHF/DSS are still
incompletely understood (Innis, B. L. 1995 in: Exotic Viral
Infections J. S. Porterfield, ed. pp. 103-146 Chapman and Hall,
London). In addition to replication in lymphoid cells, it has
become evident that the liver is involved in DEN infection of
humans. Transient elevations in serum alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) levels are observed in
the majority of DEN virus-infected patients and hepatomegaly is
observed in some patients (Kalayanarooj, S. et al. 1997 J Infect
Dis 176:313-21; Kuo, C. H. et al. 1992 Am J Trop Med Hyg 47:265-70;
Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S. F. et al.
2000 Southeast Asian J Trop Med Public Health 31:259-63). DEN virus
antigen-positive hepatocytes are seen surrounding areas of necrosis
in the liver of fatal cases (Couvelard, A. et al. 1999 Hum Pathol
30:1106-10; Huerre, M. R. et al. 2001 Virchows Arch 438:107-15),
from which dengue virus sequences were identified using RT-PCR
(Rosen, L. et al. 1999 Am J Trop Med Hyg 61:720-4). Of potential
importance to the etiology of severe dengue virus infection, three
studies have demonstrated that the mean levels of serum ALT and AST
were significantly increased in patients with DHF/DSS compared to
those with DF (Kalayanarooj, S. et al. 1997 J Infect Dis
176:313-21; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S.
F. et al. 2000 Southeast Asian J Trop Med Public Health 31:259-63).
As expected, elevation of serum liver enzymes has previously been
observed in clinical trials of DEN virus vaccine candidates
(Example 8; Eckels, K. H. et al. 1984 Am J Trop Med Hyg 33:684-9;
Edelman, R. et al. 1994 J Infect Dis 170:1448-55; Kanesa-thasan, N.
et al. 2001 Vaccine 19:3179-3188; Vaughn, D. W. et al. 1996 Vaccine
14:329-36).
Based on the increasing disease burden associated with DEN virus
infection over the past several decades, a vaccine which confers
protection against the four dengue virus serotypes is needed, but
none is presently licensed. Because of the increased risk for
severe DHF/DSS associated with secondary infection with a
heterologous DEN virus serotype (Burke, D. S. et al. 1988 Am J Trop
Med Hyg 38:172-80; Halstead, S. B. et al. 1977 J Exp Med
146:218-29; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-72), an
effective vaccine must confer simultaneous protection against each
of the four DEN virus serotypes. Several approaches are presently
being pursued to develop a tetravalent vaccine against the dengue
viruses (Bancroft, W. H. et al. 1984 J Infect Dis 149:1005-10;
Bhamarapravati, N. & Sutee, Y. 2000 Vaccine 18:44-7; Butrapet,
S. et al. 2000 J Virol 74:3011-9; Guirakhoo, F. et al. 2000 J Virol
74:5477-85; Huang, C. Y. et al. 2000 J Virol 74:3020-8;
Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188). One such
approach, a live-attenuated DEN4 vaccine candidate, termed
2A.DELTA.30, was both attenuated and immunogenic in a cohort of 20
volunteers (Example 8). The recombinant 2A.DELTA.30 virus contains
a 30 nt deletion in the 3' UTR which removes nucleotides
10,478-10,507 and was found to produce a low or undetectable level
of viremia in vaccinees at a dose of 10.sup.5 PFU/vaccinee. An
asymptomatic rash was reported in 50% of volunteers, and the only
laboratory abnormality observed was an asymptomatic, transient rise
in the serum ALT level in 5 of the 20 vaccinees. All 2A.DELTA.30
vaccinees developed serum neutralizing antibodies against DEN4
virus (mean titer: 1:580), and 2A.DELTA.30 was not transmitted to
mosquitoes that fed experimentally on vaccinees (Troyer, J. M. et
al. 2001 Am J Trop Med Hyg 65:414-9). Because of the desirable
properties conferred by the .DELTA.30 mutation, chimeric vaccine
candidates are being constructed which contain the structural genes
of DEN virus type 1, 2, and 3, in the attenuated DEN4 background
bearing the genetically stable .DELTA.30 mutation. Attenuating
mutations outside of the structural genes are particularly
attractive for inclusion in antigenic chimeric vaccine candidates
because they will not affect the infectivity or immunogenicity
conferred by the major mediator of humoral immunity to DEN viruses,
the envelope (E) protein.
The presence of rash and elevated ALT levels suggests that the
2A.DELTA.30 vaccine candidate may be slightly under-attenuated in
humans. Similarly, many previous attempts to develop live
attenuated dengue virus vaccines have yielded vaccine candidates
that were either over- or under-attenuated in humans, some of which
also induced elevation of serum ALT and AST levels (Bhamarapravati,
N. & Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever D.
J. Gubler & G. Kuno eds. pp. 367-377 CAB International, New
York; Eckels, K. H. et al. 1984 Am J Trop Med Hyg 33:684-9; Innis,
B. L. et al. 1988 J Infect Dis 158:876-80; Kanesa-thasan, N. et al.
2001 Vaccine 19:3179-3188; McKee, K. T., Jr. et al. 1987 Am J Trop
Med Hyg 36:435-42). Therefore, we have developed a menu of point
mutations conferring temperature-sensitive (ts), small-plaque (sp),
and attenuation (att) phenotypes capable of attenuating DEN4
viruses to a varying degree (Example 1). We have previously
described 20 mutant viruses that exhibit a ts, but not sp,
phenotype in Vero cells or HuH-7 liver cells and that show
attenuated replication in mouse brain (Example 1). Addition of such
mutations to 2A.DELTA.30 or to other dengue virus vaccine
candidates is envisioned as yielding vaccine candidates that
exhibit a more satisfactory balance between attenuation and
immunogenicity.
In the present Example, we have extended our analysis of the panel
of 1,248 DEN4 virus clones previously generated by mutagenesis with
5-fluorouracil (5-FU) (Example 1), by identifying a set of 22 sp
mutant viruses, some of which also have a ts phenotype. Small
plaque mutant viruses were sought since such viruses are often
attenuated in humans (Bhamarapravati, N. & Yoksan, S. 1997 in:
Dengue and Dengue Hemorrhagic Fever D. J. Gubler & G. Kuno eds.
pp. 367-377 CAB International, New York; Butrapet, S. et al. 2000 J
Virol 74:3011-9; Crowe, J. E. Jr. et al. 1994 Vaccine 12:783-790;
Crowe, J. E. Jr. et al. 1994 Vaccine 12:691-699; Eckels, K. H. et
al. 1980 Infect Immun 27:175-80; Innis, B. L. et al. 1988 J Infect
Dis 158:876-80; Murphy, B. R. & Chanock, R. M. 2001 in: Fields
Virology D. M. Knipe, et al. Eds. Vol. 1, pp. 435-468 Lippincott
Williams & Wilkins, Philadelphia; Takemoto, K. K. 1966 Prog Med
Virol 8:314-48). Because natural infection with dengue viruses and
vaccination with 2A.DELTA.30 may be associated with liver toxicity
in humans, we identified mutant viruses with restricted replication
in human liver cells. Accordingly, viruses were screened for plaque
size and temperature-sensitivity in the human hepatoma cell line,
HuH-7, as well as in Vero cells. Here we describe the ts phenotype,
nucleotide sequence, and growth properties in suckling mice of 22
sp DEN4 mutant virus clones.
Cells and viruses. WHO Vero cells (African green monkey kidney
cells) and HuH-7 cells (human hepatoma cells) (Nakabayashi, H. et
al. 1982 Cancer Res 42:3858-63) were maintained as described in
Example 1. DEN4 2A virus is a wild type virus derived from a cDNA
clone of DEN4 strain 814669 (Dominica, 1981) (Lai, C. J. et al.
1991 PNAS USA 88:5139-43; Mackow, E. et al. 1987 Virology
159:217-28). The nucleotide sequence of DEN4 2A, the parent of the
5-FU mutant viruses, was previously assigned GenBank accession
number AF375822 (Example 1). The DEN4 vaccine candidate,
2A.DELTA.30, (Example 8) contains a 30 nt deletion in the 3'
untranslated region (UTR) which removes nucleotides 10,478-10,507
(Men, R. et al. 1996 J Virol 70:3930-7). The cDNA clones p4, a
modified derivative of the DEN4 2A cDNA clone, and p4.DELTA.30 were
used to generate recombinant wild type and attenuated viruses,
rDEN4 and rDEN4.DELTA.30, respectively (Example 8). GenBank
accession numbers were previously assigned as follows (virus:
accession number): DEN4 strain 814669: AF326573; 2A.DELTA.30:
AF326826; rDEN4: AF326825; rDEN4.DELTA.30: AF326827.
Generation and biological cloning of mutant viruses with a sp
phenotype. The generation of 1,248 virus clones from a pool of
5-fluorouracil-mutagenized DEN4 2A has been previously described
(Example 1). Briefly, monolayers of Vero cells were infected with
DEN4 2A at a multiplicity of infection (MOI) of 0.01 and overlaid
with MEM supplemented with 2% FBS and 1 mM 5-fluorouracil (5-FU)
(Sigma, St. Louis, Mo.), which reduced replication of DEN4 2A
100-fold. Vero cells in 96-well plates were inoculated with the
5-FU treated virus suspension, and virus clones were harvested from
plates receiving terminally-diluted virus. A total of 1,248 virus
clones were generated from the cultures treated with 1 mM 5-FU. Two
virus clones, 2A-1 and 2A-13, were generated in the same manner
from control cultures not treated with 5-FU and served as
parallel-passaged control viruses with a wild type phenotype.
Evaluation of in vitro plaque size and temperature sensitivity. The
1,248 5-FU-mutagenized virus clones were screened for temperature
sensitivity by assessing virus replication at 35.degree. C.
(permissive temperature) and 39.degree. C. (restrictive
temperature) in Vero and HuH-7 cells. Cell monolayers in 96-well
plates were inoculated with serial ten-fold dilutions of virus and
replicate plates were incubated at 35.degree. C. and 39.degree. C.
for five days in temperature-controlled water baths. Virus
replication was determined by immunoperoxidase staining as
previously described (Example 1). A collection of 193 5-FU virus
clones demonstrated a 100-fold or greater reduction in titer at
39.degree. C. in either cell line, and these presumptive ts viruses
were further characterized. The efficiency of plaque formation
(EOP) at permissive and restrictive temperatures and the plaque
size of each of the 193 virus clones were determined as follows.
Serial ten-fold dilutions of virus suspension were inoculated onto
confluent Vero cell and HuH-7 cell monolayers in replicate 24-well
plates. After incubation at 35.degree. C. for two hours, monolayers
were overlaid with 0.8% methylcellulose (EM Science, Gibbstown,
N.J.) in L-15 medium (Quality Biologicals, Gaithersburg, Md.)
supplemented with 2% FBS, gentamicin, and L-glutamine. After
incubation of replicate plates for five days at 35, 37, 38, or
39.degree. C. in temperature-controlled water baths, plaques were
visualized by immunoperoxidase staining and counted as previously
described. Plaque size of each of the 193 viruses was evaluated at
the permissive temperature (35.degree. C.) and compared to that of
DEN4 2A-13 parallel-passaged control virus with a wild type plaque
size. Mutant viruses incubated at the permissive temperature of
35.degree. C. which had a plaque size .ltoreq.1 mm or .ltoreq.0.4
mm (approximately .ltoreq.50% the size of wild type DEN4 2A-13) in
Vero or HuH-7 cells, respectively, were designated as having a sp
phenotype. The level of temperature sensitivity and plaque size of
each virus was confirmed in at least two separate experiments.
Seventy-five viruses which were confirmed to have a putative ts
and/or sp phenotype were biologically cloned an additional two
times and phenotypes were re-assessed. Twenty-two of the 75
terminally diluted viruses were found to have a sp phenotype.
Sixteen of the 22 sp mutant viruses were also found to have a ts
phenotype as defined by a 2.5 or 3.5 log.sub.10PFU/ml reduction in
virus titer in Vero or HuH-7 cells, respectively, at restrictive
temperature compared to the permissive temperature of 35.degree. C.
as previously described (Example 1). Twenty of the 75
terminally-diluted viruses were found to have a ts phenotype
without a sp phenotype and were previously described (Example 1).
The remainder of the 75 viruses did not meet either criteria for a
ts or sp mutant virus.
Evaluation of sp mutant viruses for restricted replication in
suckling mice Animal experiments were carried out in accordance
with the regulations and guidelines of the National Institutes of
Health, Bethesda, Md. Growth of DEN4 5-FU mutant viruses was
determined in Swiss Webster suckling mice (Taconic Farms,
Germantown, N.Y.). Groups of six seven-day-old mice were inoculated
intracerebrally with 10.sup.4 PFU of virus in 30 .mu.l Opti-MEM I
(Invitrogen) and the brain of each mouse was removed five days
later and individually analyzed as previously described (Example
1). Clarified supernatants of 10% suspensions of mouse brain were
frozen at -70.degree. C., and the virus titer was determined by
plaque assay in Vero cells.
Determination of the complete genomic sequence of the sp mutant
viruses. The nucleotide sequence of the 5-FU-mutagenized DEN4
viruses was determined as described in Example 8. Briefly, genomic
RNA was isolated from virus clones and cDNA was prepared by reverse
transcription and served as template for the generation of
overlapping PCR fragments. A panel of primers was designed to
sequence both strands of the PCR product from which consensus
sequences were assembled and analyzed. The nucleotide sequence of
the 5' and 3' regions of the virus genome was determined after
circularization of the RNA genome as described in Example 8.
Identification of DEN45-fluorouracil mutant viruses with a sp
phenotype. The generation of a panel of 1,248 virus clones from a
wild type DEN4 2A virus suspension mutagenized by 5-FU has been
described previously (Example 1). In the present study twenty-two
mutant viruses with a sp phenotype were identified. The plaque size
of representative mutant viruses is illustrated in FIG. 3. The
plaque size of DEN4 2A-13 virus (a parallel-passaged virus with a
wild type phenotype derived from control cultures not treated with
5-FU) was consistently smaller in HuH-7 cells than that observed in
Vero cells (FIG. 3A). Mutant viruses #569 and #1189 (FIG. 3B) were
sp in both Vero and HuH-7 cells. In contrast, 5-FU mutant virus
clones #311 and #1083 (FIG. 3C) were sp in only HuH-7 cells,
suggesting a liver cell-specific defect in replication within this
phenotypic group. As indicated in Table 7, five mutant viruses were
found to have a sp phenotype in both Vero and HuH-7 cells while 17
viruses had a sp phenotype in only HuH-7 cells. Each 5-FU mutant
virus clone was compared for a sp or ts phenotype with three
control viruses, 2A-13, wild type rDEN4, and rDEN4.DELTA.30. The
recombinant viruses, rDEN4 and rDEN4.DELTA.30, each had a plaque
size in Vero and HuH-7 cells similar to that of DEN4 2A-13
indicating that the .DELTA.30 mutation does not confer a sp
phenotype (Table 7).
Most of the sp 5-FU mutant viruses also had a ts phenotype in Vero
and/or HuH-7 cells (Table 7) since mutant viruses were initially
screened for temperature sensitivity. Temperature-sensitivity was
defined as a 2.5 or 3.5 log.sub.10PFU/ml reduction in virus titer
in Vero or HuH-7 cells, respectively, at restrictive temperature
compared to the permissive temperature of 35.degree. C. as
previously defined (Example 1). Three mutant viruses (#574, #1269
and #1189) were sp and ts in both Vero and HuH-7 cells, while nine
mutant viruses (#506-326 in Table 7) were found to be ts in both
cell types but sp only in HuH-7 cells. Four viruses (#1104, 952,
738, and 1083) were found to have a wild type phenotype in Vero
cells but were both sp and ts in HuH-7 cells. These four mutant
viruses each had a 6,000- to 600,000-fold reduction in virus titer
at 39.degree. C. in HuH-7 cells with only a 6- to 40-fold reduction
at 39.degree. C. in Vero cells. Finally, sp mutant viruses were
identified which did not have a ts phenotype in either cell line;
two of these viruses (#569 and #761) were sp in both Vero and HuH-7
cells and four viruses (#1096-1012) were sp in only HuH-7 cells
(Table 7). As described previously, the .DELTA.30 mutation did not
confer temperature-sensitivity in either cell line (Example 1).
The sp 5-FU mutant viruses have restricted replication in suckling
mouse brain. The 22 sp DEN4 5-FU mutant viruses were evaluated for
their ability to replicate in the brain of one-week-old suckling
mice. As a marker for in vivo attenuation, their level of
replication was compared with that of the parallel-passaged control
virus with a wild type phenotype, 2A-13 (Table 7). Nineteen of 22
sp mutant viruses had a greater than 100-fold reduction in virus
replication in the brain of suckling mice compared to 2A-13 and
nine viruses had a reduction of greater than 10,000-fold.
The five mutant viruses which were sp in both Vero and HuH-7 cells
were 5,000-fold to 100,000-fold restricted in replication compared
to 2A-13. Two of these mutant viruses, #569 and #761, were not ts
in either cell line but had a reduction in virus titer of greater
than 10,000-fold in mouse brain, indicating that the sp phenotype
in both Vero and HuH-7 cells is an important surrogate marker for
attenuated replication in suckling mouse brain. 5-FU mutant viruses
which were sp in only HuH-7 cells had a more variable range of
replication in mouse brain. Three viruses had a mean reduction in
virus titer of less than 10-fold when compared to 2A-13 virus.
However, 8 of 13 viruses which were ts in Vero and/or HuH-7 cells
but sp in only HuH-7 cells had a greater than 5,000-fold reduction
in virus replication. The results of the in vivo replication
analysis of the previously described 20 ts 5-FU mutant viruses
(Example 1) and the 22 sp mutant viruses are summarized in Table 8.
Mutant viruses with both a sp and ts phenotype were found to have a
significantly greater level of attenuation in the brain of suckling
mice when compared to viruses with only a ts phenotype.
Sequence analysis of the sp 5-FU mutant viruses. To initiate an
analysis of the genetic basis of the ts, sp, or att phenotype of
the 22 sp mutant viruses, the complete nucleotide sequence of each
virus genome was determined and is summarized in Table 9 (sp in
Vero and HuH-7 cells) and Table 10 (sp in only HuH-7 cells). All
identified mutations were nucleotide substitutions, as deletions or
insertions were not observed. Point mutations were distributed
throughout the genome, including the 3' UTR as well as in all
coding regions. Because all 5-FU mutant viruses were found to have
at least two mutations (two to six), the observed phenotypes cannot
be directly attributed to a specific mutation. The majority of sp
viruses also contained translationally silent point mutations (none
to four) in the structural or non-structural coding regions.
However, these silent mutations are not expected to contribute to
the observed phenotypes. Six of the 22 sp mutant viruses (Tables 9
and 10) were found to have mutations in only the NS genes and/or
the 3' UTR, indicating that the sp phenotype can be conferred by
mutations outside of the structural genes.
Presence of identical mutations in multiple 5-FU mutant viruses.
Analysis of the complete nucleotide sequence data for the 5-FU
mutant viruses identified several repeated mutations which were
present in two or more viruses. Such mutations were also identified
previously during our analysis of twenty 5-FU mutant viruses with a
ts but not sp phenotype (Example 1). Because these mutations
occurred in viruses together with additional mutations, the
contribution of the repeated mutations to the observed sp, ts, and
att phenotypes remains empirical. Table 11 lists the repeated
mutations found among the 20 ts (not sp) mutant viruses described
previously (Example 1) and the 22 sp mutant viruses described here.
Repeated mutations were identified in the following genes: two in
E, two in NS3, five in NS4B, one in NS5, and two in the 3' UTR.
Interestingly, within a thirty nucleotide region of NS4B (nt
7153-7182), there were five different nucleotide substitutions
which were found in sixteen viruses. Also at nt 7,546 in NS4B, an
amino acid substitution (Ala.fwdarw.Val) was found in 10 different
5-FU mutant viruses. The significance of these repeated mutations
in NS4B as well as in other DEN4 genomic regions remains empirical,
but a reasonable explanation for this phenomenon is that these
mutations are involved in adaptation of DEN4 virus for efficient
growth in Vero cells, as further discussed in Example 6.
Discussion. As part of a molecular genetic vaccine strategy, we
have developed attenuating mutations that are envisioned as being
useful in the development of a live attenuated tetravalent dengue
virus vaccine. Specifically, mutations which restrict replication
of the vaccine virus in human liver cells were generated since
there was some residual virulence of the rDEN4.DELTA.30 vaccine
candidate for the liver of humans. Mutant viruses with a sp
phenotype were sought in both Vero cells and HuH-7 human liver
cells, in order to identify host-range mutant viruses that were
specifically restricted in replication in HuH-7 cells (sp in HuH-7
but not in Vero). Such mutations are envisioned as being useful in
limiting replication of a candidate vaccine in the liver of
vaccinees while preserving both efficient replication in Vero cells
and immunogenicity in vivo.
Several observations from the present study indicate that sp
mutations confer an att phenotype in vivo. This is not surprising
since attenuation in suckling mouse brain has been reported for
live DEN virus vaccine candidates possessing sp phenotypes,
including the DEN2 PDK-53 and DEN2 PR-159/S-1 vaccine strains
(Bhamarapravati, N. & Yoksan, S. 1997 in: Dengue and Dengue
Hemorrhagic Fever D. J. Gubler & G. Kuno eds. pp. 367-377 CAB
International, New York; Butrapet, S. et al. 2000 J Virol
74:3011-9; Eckels, K. H. et al. 1980 Infect Immun 27:175-80; Innis,
B. L. et al. 1988 J Infect Dis 158:876-80). Each of 22 DEN4 5-FU
mutant viruses with a sp phenotype (some of which were also ts) in
either Vero or HuH-7 cells manifested restricted replication in the
brains of mice. Six 5-FU mutant viruses with a sp phenotype in the
absence of a ts phenotype were more attenuated in the brains of
suckling mice than mutant viruses with solely a ts phenotype
(Example 1), indicating that the sp phenotype specifies a greater
level of attenuation for mouse brain than does the ts phenotype.
Mutant viruses with both a ts and sp phenotype had an even greater
reduction in replication, further indicating that the attenuation
conferred by the ts and sp phenotypes can be additive. Importantly,
seventeen of the 22 sp mutant viruses were host-range sp mutant
viruses, being sp only in HuH-7 cells. Since such mutations are
envisioned as being useful in restricting the replication of a DEN4
virus in human liver cells, we used nucleotide sequence analysis to
determine the genetic basis of the sp phenotype.
Analysis of the complete genomic sequence of the 22 sp DEN4 viruses
revealed substitutions in the 3' UTR as well as coding mutations in
all genes except NS4A. It was first noted that several specific
mutations were present in two or more of the 22 sp DEN4 mutant
viruses and that many of these same mutations were also previously
identified among the set of 20 ts DEN4 mutant viruses (Example 1).
Since flaviviruses can rapidly accumulate mutations during passage
in tissue culture (Dunster, L. M. et al. 1999 Virology 261:309-18;
Mandl, C. W. et al. 2001 J Virol 75:5627-37), many of these
over-represented mutations, previously referred to as putative Vero
cell adaptation mutations (Example 1), likely promote efficient
replication in Vero cells and were selected unintentionally during
the biological cloning of the mutant viruses. The effect of these
mutations on DEN virus replication in Vero cells, the proposed
substrate for vaccine manufacture, is discussed in Example 6.
The sp mutations identified among the 5-FU mutant viruses are
envisioned as being useful in several different approaches for the
development of DEN virus vaccine strains. As described above for
the generation of antigenic chimeric viruses, one or more sp
attenuating mutations are envisioned as being added to the
attenuated DEN4.DELTA.30 genetic background to supplement the att
phenotype of the .DELTA.30 mutation. A second approach is to
introduce a sp attenuating mutation, with or without .DELTA.30,
into infectious cDNA clones of the other three DEN serotypes. The
ability to transfer mutations among genetically-related viruses and
maintain similar att phenotypes has been previously demonstrated
(Skiadopoulos, M. H. et al. 1999 Virology 260:125-35). These
distinct strategies are envisioned as being useful as separate or
complementary approaches to the construction of a tetravalent DEN
virus vaccine, underlining the importance of the identification of
a large panel of att mutations within the DEN viruses.
Example 3
Recombinant DEN4 Viruses Containing Mutations Identified in 5-FU
Mutant Viruses Show Restricted Replication in Suckling Mouse Brain
and in SCID Mice Transplanted with Human Liver Cells
Data was presented, in Examples 1 and 2 that summarizes the
generation, characterization and sequence analysis of 42 attenuated
mutant DEN4 viruses. For three of the mutant viruses (#239, 489,
and 773) with a single missense mutation at nt position 4995 in
NS3, it was clear that the identified mutation specified the ts and
att phenotypes. This conclusion was confirmed in Example 1 by
tissue culture and in vivo characterization of rDEN4-4995, a
recombinant virus into which the 4995 mutation had been introduced
by site-directed mutagenesis. In this analysis, rDEN4-4995
exhibited the same level of temperature sensitivity and attenuation
as 5-FU mutant viruses #239, 489, and 773. The individual
mutation(s) in the remaining 5-FU mutant viruses that specify the
observed phenotypes remains to be identified, since most of these
viruses possess more than one nucleotide substitution. We have
conducted an analysis to identify the mutations in a subset of the
other 39 mutant viruses that specify the ts, sp, and att phenotypes
by introduction of each mutation into the wt DEN4 cDNA (p4) and
evaluation of the phenotypes of the resulting recombinant DEN4
viruses bearing the individual mutations. Previous studies of a
DEN2 virus vaccine candidate (Butrapet, S. et al. 2000 J Virol
74:3011-9) as well as other virus vaccines (Whitehead, S. S. et al.
1999 J Virol 73:871-7) have demonstrated the utility of this
approach for the identification of the genetic basis of
attenuation.
As described in Examples 1 and 2, 19 5-FU mutant viruses were
identified which were found to contain coding mutations in only the
NS genes and/or nucleotide substitutions in the 5' or 3' UTR which
would facilitate the generation of antigenic chimeric viruses. In
the present example, the genetic basis of the observed sp, ts, and
mouse brain att phenotypes was identified for these 19 viruses
using reverse genetics to generate recombinant DEN4 (rDEN4) viruses
containing individual mutations identified in the panel of DEN4
mutant viruses. In addition, the 19 5-FU mutant viruses were
evaluated for replication in a novel small animal model for DEN4
virus replication, SCID mice transplanted with HuH-7 cells
(SCID-HuH-7), and the genetic basis of the att viruses was
identified using mutant rDEN4 viruses. Also presented are findings
describing the generation and characterization of a recombinant
virus containing two of the identified attenuating mutations as
well as combination of select 5-FU mutations with the .DELTA.30
mutation.
Generation of rDEN4 viruses containing 5-FU mutations. The methods
used for the generation of rDEN4 viruses are outlined in FIG. 4 and
are similar to those described in Example 1. Briefly, the p4 cDNA
was digested with the appropriate restriction enzymes and the
resulting fragments were subcloned into a modified pUC119 vector.
For Kunkel mutagenesis, single-stranded DNA preparations of the
pUC-NS vectors were made, and primers were designed to individually
introduce mutations that were present in the 5-FU mutant viruses.
The sequences of the 41 mutagenic oligonucleotides used to generate
the single-mutation recombinant viruses are presented in Table 12.
Primers were designed to co-introduce or co-ablate a
translationally-silent restriction enzyme site in the cDNA, which
greatly facilitates the screening and identification of cDNA clones
possessing the mutant sequence. Fragments containing the introduced
mutations were cloned back into p4, and nucleotide sequence
analysis confirmed the presence of the nucleotide changes. A total
of 33 rDEN4 viruses was generated which contained each of the
individual mutations present in the 19 5-FU mutant viruses
containing only coding mutations in the NS genes and/or nucleotide
substitutions in the 5' or 3' UTR. An additional 8 rDEN4 viruses
were generated from mutations identified in the remaining panel of
42 5-FU mutant viruses.
A cDNA clone was also generated which combined the mutations
identified at nt position 4995 in NS3 and 7849 in NS5. The 7849
mutation was introduced into the p4-4995 cDNA clone by replacing
the XmaI-PstI fragment with that derived from the p4-7849 cDNA
clone. The presence of both mutations was confirmed by sequence
analysis. The .DELTA.30 mutation was introduced into the 3' UTR of
the individual mutant cDNA clones by replacing the MluI-KpnI
fragment with that derived from the p4.DELTA.30 cDNA clone, and the
presence of the deletion was confirmed by sequence analysis.
Recombinant viruses were recovered by transfection of Vero or C6/36
cells with RNA transcripts derived from the mutant cDNA clones as
described in Example 1. Recovered viruses were terminally diluted
twice and working stocks of viruses were prepared in Vero cells.
Each of the mutant cDNA clones was recovered after transfection as
expected since the 5-FU mutant viruses containing these mutations
were viable.
Characterization of ts and att phenotypes of the rDEN4 viruses
containing introduced mutations. Of the 19 5-FU mutant viruses with
mutations in only NS genes and/or the 5' or 3' UTR, six had an sp
phenotype (Table 13), ten had a ts phenotype in Vero and HuH-7
cells (Table 14), and three had a ts phenotype in only HuH-7 cells
(Table 15). For the six sp 5-FU mutant viruses, #738, 922, 1081,
1083, 1136, and 1189, seventeen mutations identified by sequence
analysis resulted in a coding change or a nucleotide change in the
UTR and each was engineered into an individual DEN4 cDNA clone.
Virus containing each defined mutation was successfully recovered
and propagated and was tested for efficiency of plaque formation in
Vero and HuH-7 cells at various temperatures, plaque size
phenotype, and growth properties in suckling mice using methods
previously described in Examples 1 and 2.
Table 13 lists the phenotypes of the six sp 5-FU mutant parent
viruses and those of the 17 rDEN4 viruses encoding single mutations
present in the parent virus. For example, 5-FU mutant #1189
(parent), which was ts and sp in both cell lines and had an almost
10,000-fold reduction in replication in suckling mouse brain,
contained 4 coding mutations at nt position 3303 in NS1, 4812 and
5097 in NS3, and 7182 in NS4B. Analysis of the four rDEN4 viruses
containing each of these mutations indicated that rDEN4-5097 had a
ts, sp, and att phenotype while rDEN4-3303, rDEN4-4812, and
rDEN4-7182 had no discernible phenotypes, indicating that the
mutation at nt 5097 was responsible for the phenotype observed in
the 5-FU parent, #1189. Thus, analysis of the relative
contributions of the four mutations present in the 5-FU mutant
#1189 to its attenuation phenotype provides the framework for a
similar analysis of the remaining 5-FU mutant viruses. This
analysis specifically demonstrates the methods used to identify
mutations contributing to the observed phenotype. The ts, sp, and
att phenotypes of 5-FU parent viruses #738, 922, 1081, and 1083,
were similarly attributed to single mutations 3540, 4306, 2650, and
10634, respectively. However, two separate mutations (3771 and
4891) contributed to the phenotypes of 5-FU mutant virus #1136.
Table 14 lists the genetic basis of the ts and mouse brain
attenuation for the ten 5-FU mutant viruses with ts phenotypes in
both Vero and HuH-7 cells. As described in Example 1, the 4995
mutation which is the only mutation present in three 5-FU mutant
viruses, #239, #489, and #773, was found to confer a ts and att
phenotype, confirming the genetic basis for the phenotypes
exhibited by these viruses. In three separate experiments, the
rDEN4-4995 virus was found to have an approximately 1,000-fold
decrease in replication in the brains of suckling mice when
compared to that of wild-type virus (Table 6 and 14). The 4995
mutation is also present in 5-FU mutant viruses #473, #759, and
#816, each of which has additional mutations. The ts and att
phenotypes observed in these viruses can be attributed to the 4995
mutation since the additional mutations did not show discernible
phenotypes. Interestingly, 5-FU mutant virus #938 has the 4995
mutation and an additional mutation at nt 3442 in NS1 with both
mutations independently conferring restricted replication in mouse
brain. The remaining three 5-FU parent viruses in Table 14, #173,
#509, and #1033, were found to each contain a single mutation
responsible for the att phenotype: 7849, 8092, and 4907,
respectively.
Three 5-FU mutant viruses, #686, #992, and #1175 with HuH-7
cell-specific ts phenotypes are listed in Table 15. Mutations in
NS3 (5695) and NS5 (10186) were found to confer the phenotypes
observed for parent virus #992 and #1175. Interestingly, two
mutations in NS2A, 3575 and 4062, were found to result in a
synergistic increase in the level of attenuation. Both individual
mutations had an approximately 100-fold decrease in virus
replication in the brain while the parent virus with both mutations
had an almost 10,000-fold reduction. Table 16 lists two additional
mutations with an att phenotype, 4896 and 6259 in NS3.
Replication of DEN4 viruses in SCID mice transplanted with HuH-7
cells. Since DEN viruses replicate poorly in the liver of mice and
corresponding studies are impractical to conduct in non-human
primates, an animal model that evaluates the in vivo level of
replication of DEN virus in liver cells was developed based on a
recent report examining the replication of DEN virus in SCID mice
transplanted with a continuous cell line of human liver tumor cells
(An, J. et al. 1999 Virology 263:70-7). SCID mice transplanted with
human continuous cell lines, primary cells, or organized tissues
have similarly been used to study the replication of other viruses
which lack a suitable small animal model (Mosier, D. E. 2000
Virology 271:215-9). In our study, SCID mice were transplanted with
HuH-7 cells since DEN4 virus replicated efficiently in these cells
in tissue culture and since these were the cells used to define the
host-range phenotype. These studies are envisioned as addressing
the utility of examining DEN virus infection in SCID
mouse-xenograft models for vaccine development (An, J. et al. 1999
Virology 263:70-7; Lin, Y. L. et al. 1998 J Virol 72:9729-37).
To further examine the in vivo growth properties of the 19 5-FU
mutant DEN4 viruses with mutations in only the NS genes and/or the
3' UTR and selected corresponding rDEN4 mutant viruses, replication
was assessed in SCID mice transplanted with HuH-7 cells
(SCID-HuH-7). For analysis of DEN4 virus replication in SCID-HuH-7
mice, four to six week-old SCID mice
(Tac:Icr:Ha(ICR)-Prkdc.sup.scid) (Taconic Farms) were injected
intraperitoneally with 10.sup.7 HuH-7 cells suspended in 200 .mu.l
phosphate-buffered saline (PBS). In preparation for
transplantation, HuH-7 cells were propagated in cell culture as
described above and harvested by trypsinization at approximately
80% confluence. Cells were washed twice in PBS, counted,
resuspended in an appropriate volume of PBS, and injected into the
peritoneum of mice. Tumors were detected in the peritoneum five to
six weeks after transplantation, and only mice with apparent tumors
were used for inoculation. Mice were infected by direct inoculation
into the tumor with 10.sup.4 PFU of virus in 50 .mu.l Opti-MEM I.
Mice were monitored daily for seven days and serum for virus
titration was obtained by tail-nicking on day 6 and 7.
Approximately 400 .mu.l blood was collected in a serum separator
tube (Sarstedt, Germany), centrifuged, and serum was aliquoted and
stored at -70.degree. C. The virus titer was determined by plaque
assay in Vero cells. Seven days after infection, most mice
developed morbidity and all mice were sacrificed. Tumors were
excised and weighed to confirm uniformity of the experimental
groups.
Preliminary experiments indicated that SCID-HuH-7 mice inoculated
with DEN4 2A-13 directly into the tumor developed viremia with
maximum levels (up to 8.0 log.sub.10PFU/ml serum) achieved on day 5
(Table 17). Virus could also be detected in brain, liver, and tumor
homogenates.
The level of viremia in SCID-HuH-7 mice infected with parental 5-FU
or rDEN4 mutant viruses was compared with that of the
parallel-passaged control virus, 2A-13, or rDEN4, respectively.
Results of 4 separate experiments indicated that the vaccine
candidate, rDEN4.DELTA.30, had an almost 10-fold reduction in virus
replication compared to wild type rDEN4 (Table 13) which reflects
the apparent attenuation of the rDEN4.DELTA.30 vaccine candidate in
humans (Example 8). Results in Tables 13 to 15 indicate that three
5-FU mutant viruses had a greater than 100-fold reduction in
viremia in the SCID-HuH-7 mice compared to wild type 2A-13 virus:
#1081, #1083, and #1189. The common phenotype among these viruses
was a sp phenotype in HuH-7 cells. Analysis of the genetic basis of
the att phenotype in these parent 5-FU mutant viruses identified
three individual mutations in NS1, NS3, and the 3' UTR which
conferred at least a 100-fold reduction in viremia. Specifically,
rDEN4-2650 (NS1), rDEN4-5097 (NS3), and rDEN4-10634 (3' UTR)
manifested a 2.2, 3.6, and 4.3 log.sub.10PFU/ml reduction in peak
titer of viremia compared to rDEN4, respectively. These mutations
also conferred the att phenotype in suckling mouse brain. 5-FU
mutant virus #738 and #509 had a reduction in viremia in the
SCID-HuH-7 mice compared to wild type 2A-13 of 1.9 and 1.5
log.sub.10PFU/ml, respectively, and the genetic basis for these
phenotypes is envisioned as being assessed on an empirical
basis.
This analysis of the genetic basis of the phenotypes specified by
the mutations in the 5-FU mutant viruses that manifested restricted
replication in SCID-HuH-7 mice indicated that (1) three separate
mutations conferred the att phenotype; (2) these mutations were
located in two proteins, NS1 and NS3, and in the 3' UTR; (3) these
three mutations were fully responsible for each of the cell culture
(ts or sp) and in vivo (attenuation in mouse brain and SCID-HuH-7
mice) phenotypes of the parent viruses; and (4) two of the three
mutations specify the host-range sp phenotype (sp on HuH-7 only)
and therefore are envisioned as being useful in a vaccine virus.
Although the relevance of such SCID-transplant models to virus
replication and disease in humans is unknown, the identification of
three novel mutations which restrict DEN4 virus replication in
SCID-HuH-7 mice is envisioned as facilitating an examination of the
correlation between the att phenotype in SCID-HuH-7 mice with that
in rhesus monkeys or humans. Such mutations, specifically the
host-range sp mutations, are envisioned as being useful in
conjunction with the .DELTA.30 or other mutation to decrease the
residual virulence of rDEN4.DELTA.30 or other dengue virus for the
human liver, and studies are envisioned as being conducted to
construct such rDEN4 viruses and evaluate them in monkeys and
humans (Example 8).
Combination of two 5-FU mutations results in an additive ts
phenotype. The ability to combine individual mutations in rDEN4
virus as a means to modulate the phenotype of the resulting double
mutant virus is a major advantage of using recombinant cDNA
technology to generate or modify dengue virus vaccine candidates.
Addition of multiple ts and att mutations to recombinant vaccine
viruses is envisioned as improving the phenotypic stability of the
double recombinant due to the decreased possibility of co-reversion
of the two mutations to wild-type virulence (Crowe, J. E. Jr. et
al. 1994a Vaccine 12:783-790; Skiadopoulos, M. H. et al. 1998 J
Virol 72:1762-8; Subbarao, E. K. et al. 1995 J Virol 69:5969-5977;
Whitehead, S. S. et al. 1999 J Virol 73:871-7). The mutations
identified at nt position 4995 in NS3 and 7849 in NS5 were combined
in a single p4 cDNA clone and a recombinant virus, designated
rDEN4-4995-7849, was recovered and evaluated for its ts and att
phenotypes (Table 18). rDEN4-4995-7849 was more ts than either
recombinant virus containing the individual mutations (Table 18),
indicating the additive effect of the two ts mutations. The
rDEN4-4995-7849 virus had a greater than 10,000-fold reduction in
replication in the brains of suckling mice. The reduction in
replication of the double mutant virus was only slightly increased
over that of rDEN4-7849, however, a difference in the level of
replication between rDEN4-4995-7849 and rDEN4-7849 would be
difficult to detect since the level of replication of both viruses
was close to the lower limit of detection (2.0 log.sub.10PFU/g
brain).
Combination of selected 5-FU mutations with the .DELTA.30 mutation
confers increased attenuation of rDEN4.DELTA.30 for the brains of
suckling mice. To define the effect of adding individual mutations
to the attenuated rDEN4.DELTA.30 background, five combinations have
been constructed: rDEN4.DELTA.30-2650, rDEN4.DELTA.30-4995,
rDEN4.DELTA.30-5097, rDEN4.DELTA.30-8092, and rDEN4.DELTA.30-10634.
Addition of such missense mutations with various ts, sp, and att
phenotypes is envisioned as serving to decrease the reactogenicity
of rDEN4.DELTA.30 while maintaining sufficient immunogenicity.
The .DELTA.30 mutation was introduced into the 3' UTR of the
individual mutant cDNA clones by replacing the MluI-KpnI fragment
with that derived from the p4.DELTA.30 cDNA clone, and the presence
of the deletion was confirmed by sequence analysis. Recombinant
viruses were recovered by transfection in C6/36 cells for each
rDEN4 virus. However, upon terminal dilution and passage, the
rDEN4.DELTA.30-5097 virus was found to not grow to a sufficient
titer in Vero cells and was not pursued further. This is an example
of a cDNA in which the 5-FU mutation and the .DELTA.30 mutation are
not compatible for efficient replication in cell culture. To begin
the process of evaluating the in vivo phenotypes of the other four
viruses which replicated efficiently in cell culture, rDEN4 viruses
containing the individual mutations and the corresponding
rDEN4.DELTA.30 combinations were tested together for levels of
replication in suckling mouse brain. The results in Table 19
indicate that addition of each of the mutations confers an
increased level of attenuation in growth upon the rDEN4.DELTA.30
virus, similar to the level conferred by the individual 5-FU
mutation. No synergistic effect in attenuation was observed between
the missense mutations and .DELTA.30. These results indicate that
the missense mutations at nucleotides 2650, 4995, 8092, and 10634
are compatible with .DELTA.30 for growth in cell culture and in
vivo and can further attenuate the rDEN4.DELTA.30 virus in mouse
brain. Further studies in SCID-HuH-7 mice, rhesus monkeys, and
humans are envisioned as establishing the effect of the combination
of individual mutations and .DELTA.30 upon attenuation and
immunogenicity (Example 8).
By identifying the specific mutations in the 5-FU mutant viruses
which confer the observed phenotypes, a menu of defined ts, sp, and
att mutations is envisioned as being assembled (see Example 7).
Numerous combinations of two or more of these mutations are
envisioned as being selected with or without the .DELTA.30
mutation. Such mutations and their combinations are envisioned as
being useful for the construction of recombinant viruses with
various levels of in vivo attenuation, thus facilitating the
generation of candidate vaccines with acceptable levels of
attenuation, immunogenicity, and genetic stability.
Example 4
Generation of DEN4 Mutant Viruses with Temperature-Sensitive and
Mouse Attenuation Phenotypes Through Charge-Cluster-to-Alanine
Mutagenesis
The previous Examples described the creation of a panel of DEN4
mutant viruses with ts, sp, and att phenotypes obtained through
5-FU mutagenesis. As indicated in these Examples, the attenuating
mutations identified in the 5-FU mutant viruses are envisioned as
having several uses including (1) fine tuning the level of
attenuation of existing dengue virus vaccine candidates and (2)
generation of new vaccine candidates by combination of two or more
of these attenuating mutations. In the current example, we created
a second panel of mutant viruses through charge-cluster-to-alanine
mutagenesis of the NS5 gene of DEN4 and examined the resulting
mutant viruses for the ts, sp, and att phenotypes as described in
Examples 1 and 2. The charge-cluster-to-alanine mutant viruses
recovered demonstrated a range of phenotypes including ts in Vero
cells alone, ts in HuH-7 cells alone, ts in both cell types, att in
suckling mouse brains, and att in SCID-HuH-7 mice.
The usefulness of mutant viruses expressing these phenotypes has
already been described, however charge-cluster-to-alanine mutant
viruses possess some additional desirable characteristics. First,
the relevant mutations are envisioned as being designed for use in
the genes encoding the non-structural proteins of DEN4, and
therefore are envisioned as being useful to attenuate DEN1, DEN2,
and DEN3 antigenic chimeric recombinants possessing a DEN4 vector
background. Second, the phenotype is usually specified by three or
more nucleotide changes, rendering the likelihood of reversion of
the mutant sequence to that of the wild type sequence less than for
a single point mutation, such as mutations identified in the panel
of 5-FU mutant viruses. Finally, charge-cluster-to-alanine
attenuating mutations are envisioned as being easily combinable
among themselves or with other attenuating mutations to modify the
attenuation phenotype of DEN4 vaccine candidates or of DEN1, DEN2,
and DEN3 antigenic chimeric recombinant viruses possessing a DEN4
vector background.
Charge-Cluster-to-Alanine-Mutagenesis. The cDNA p4, from which
recombinant wild type and mutant viruses were generated, has been
described in Examples 1, 2, and 3 and in FIG. 4.
Charge-cluster-to-alanine mutagenesis (Muylaert, I. R. et al. 1997
J Virol 71:291-8), in which pairs of charged amino acids are
replaced with alanine residues, was used to individually mutagenize
the coding sequence for 80 pairs of contiguous charged amino acids
in the DEN4 NS5 gene. Subclones suitable for mutagenesis were
derived from the full length DEN4 plasmid (p4) by digestion with
XmaI/PstI (pNS5A), PstI/SacII (pNS5B) or SacII/MluI (pNS5C) at the
nucleotide positions indicated in FIG. 4. These fragments were then
subcloned and Kunkel mutagenesis was conducted as described in
Examples 1 and 3. To create each mutation, oligonucleotides were
designed to change the sequence of individual pairs of codons to
GCAGCX (SEQ ID NO: 69), thereby replacing them with two alanine
codons (GCX) and also creating a BbvI restriction site (GCAGC) (SEQ
ID NO: 70). The BbvI site was added to facilitate screening of
cDNAs and recombinant viruses for the presence of the mutant
sequence. Restriction enzyme fragments bearing the alanine
mutations were cloned back into the full-length p4 plasmid as
described in Examples 1 and 3.
Initial evaluation of the phenotype of the 32
charge-cluster-to-alanine mutant viruses revealed a range in
restriction of replication in suckling mouse brain and SCID-HuH-7
mice. To determine whether attenuation could be enhanced by
combining mutations, double mutant viruses carrying two pairs of
charge-cluster-to-alanine mutations were created by swapping
appropriate fragments carrying one pair of mutations into a
previously-mutagenized p4 cDNA carrying a second pair of mutations
in a different fragment using conventional cloning techniques.
Transcription and Transfection. 5'-capped transcripts were
synthesized in vitro from mutagenized cDNA templates using AmpliCap
SP6 RNA polymerase (Epicentre, Madison, Wis.). Transfection
mixtures, consisting of 1 .mu.g of transcript in 60 .mu.l of
HEPES/saline plus 12 .mu.l of dioleoyl trimethylammonium propane
(DOTAP) (Roche Diagnostics Corp., Indianapolis, Ind.), were added,
along with 1 ml Virus production-serum free medium (VP-SFM) to
subconfluent monolayers of Vero cells in 6-well plates. Transfected
monolayers were incubated at 35.degree. C. for approximately 18 hr,
cell culture medium was removed and replaced with 2 ml VP-SFM, and
cell monolayers were incubated at 35.degree. C. After 5 to 6 days,
cell culture medium was collected, and the presence of virus was
determined by titration in Vero cells followed by immunoperoxidase
staining as previously described. Recovered virus was amplified by
an additional passage in Vero cells, and virus suspensions were
combined with SPG (sucrose-phosphate-glutamate) stabilizer (final
concentration: 218 mM sucrose, 6 mM L-glutamic acid, 3.8 mM
potassium phosphate, monobasic, and 7.2 mM potassium phosphate,
dibasic, pH 7.2), aliquoted, frozen on dry ice, and stored at
-70.degree. C.
cDNA constructs not yielding virus after transfection of Vero cells
were used to transfect C6/36 cells as follows. Transfection
mixtures, as described above, were added, along with 1 ml of MEM
containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 2 mM
non-essential amino acids, and 0.05 mg/ml gentamicin, to monolayers
of C6/36 cells. Transfected cell monolayers were incubated at
32.degree. C. for 18 hr, cell culture medium was removed and
replaced with 2 ml fresh medium, and cell monolayers were incubated
at 32.degree. C. After 5 to 6 days, cell culture media were then
used to infect Vero cells and incubated for 5-6 days, at which time
cell culture media were collected, frozen and titered as described
above.
Recovered viruses were biologically cloned by two rounds of
terminal dilution in Vero cells followed by an additional
amplification in Vero cells. Briefly, virus was initially diluted
to a concentration of approximately 20 PFU/ml in VP-SFM and then
subjected to a series of two-fold dilutions across a 96-well plate.
Virus dilutions were used to infect Vero cell monolayers in a
96-well plate and incubated for 5 to 6 days at 35.degree. C.
Following incubation, cell culture media were removed and
temporarily stored at 4.degree. C., and the virus-positive cell
monolayers were identified by immunoperoxidase staining. Terminal
dilution was achieved when .ltoreq.25% of cell monolayers were
positive for virus. Cell culture medium from a positive monolayer
at the terminal dilution was subjected to an additional round of
terminal dilution. Following the second terminal dilution, virus
was amplified in Vero cells (75 cm.sup.2 flask), collected and
frozen as previously described.
Assays for temperature-sensitivity and mouse attenuation. Assay of
the level of temperature sensitivity of the
charge-cluster-to-alanine mutant viruses in Vero and HuH-7 cells
and their level of replication in the brain of suckling mice were
conducted as described in Example 1 and assay of the level of
replication in SCID-HuH-7 mice was conducted as described in
Example 3.
Charge-cluster-to-alanine mutant viruses are viable and show
temperature-sensitive and mouse attenuation phenotypes. Of 80
full-length DEN4 cDNA constructs containing a single pair of
charge-to-alanine mutations, virus was recovered from 32 in either
Vero or C6/36 cells (FIG. 5). The level of temperature sensitivity
of wt rDEN4, rDEN4.DELTA.30, and the 32 mutant viruses is
summarized in Table 20. One mutant virus (645-646) was ts in Vero
but not HuH-7 cells and 7 mutant viruses were ts in HuH-7 but not
Vero cells. Such mutants whose temperature sensitivity is host-cell
dependent are referred to as temperature-sensitive, host-range
(tshr) mutants. Thirteen mutant viruses were ts in both cell types,
and 11 mutant viruses were not ts on either cell type. Thus a total
of 21 mutant viruses were ts with 8 mutant viruses exhibiting an
tshr specificity. None of the mutant viruses showed a small plaque
phenotype at permissive temperature. Mutant viruses showed a wide
range (0 to 10,000-fold) of restricted replication in suckling
mouse brain (Table 20). Fourteen mutant viruses were attenuated in
suckling mouse brain, arbitrarily defined as a .gtoreq.1.5
log.sub.10-unit reduction in virus titer. There was no correlation
between attenuation in mouse brain and temperature sensitivity in
either Vero cells (Kendall Rank correlation: P=0.77) or HuH-7 cells
(Kendall Rank correlation: P=0.06).
Thirteen mutant viruses that either showed an att phenotype in
suckling mouse brain or whose unmutated charged amino acid pair was
highly conserved among the four DEN serotypes (see Example 7) were
assayed for att in SCID-HuH-7 mice (Table 21). Three of these
mutant viruses showed>100-fold decrease in replication relative
to wild type DEN4. Overall, mean log reduction from wild type in
suckling mice did not show significant correlation with mean log
reduction in SCID-HuH-7 mice (Spearman rank correlation, N=13,
P=0.06). However, mutant virus 200-201 was unusual in that it
showed a high level of restriction in SCID-HuH-7 mice but little
restriction in suckling mouse brain. When virus 200-201 was removed
from the analysis, restriction of replication in suckling and
SCID-HuH-7 mice showed a significant correlation (Spearman rank
correlation, N=12, P=0.02).
Combining charge-cluster-to-alanine mutations present in two
viruses into one virus can enhance its ts and att phenotypes. Six
paired mutations were combined into fourteen double-pair mutant
viruses, of which six could be recovered in Vero or C6/36 cells
(Table 22). All of the individual paired mutations used in
double-pair mutant viruses were ts on HuH-7 cells, none was ts in
Vero cells, and for all combinations at least one mutation pair
conferred an att phenotype in suckling mouse brain. Evaluation of
four of the double-pair mutant viruses (Table 23) revealed that
combining charge-cluster-to-alanine mutation pairs invariably
resulted in the acquisition of a ts phenotype in Vero cells (4 out
of 4 viruses) and often resulted in a lowered shutoff temperature
in HuH-7 cells (3 out of 4 viruses). In half of the viruses
assayed, combination of charge-cluster-to-alanine mutation pairs
resulted in enhanced restriction of replication (10-fold greater
than either component mutation) in suckling mouse brain (Table 23)
and in SCID-HuH-7 mice (Table 24).
Summary. The major usefulness of the charge-cluster-to-alanine
mutations stems from their design: they are located in the DEN4
non-structural gene region and therefore are envisioned as being
useful to attenuate DEN4 itself as well as antigenic chimeric
viruses possessing the DEN4 NS gene region. Furthermore, they are
predicted to be phenotypically more stable than the
single-nucleotide substitution mutant viruses such as the 5-FU
mutant viruses. Finally, combinations of mutations are envisioned
as being created in order to fine-tune attenuation and to further
stabilize attenuation phenotypes.
Example 5
Identification and Characterization of DEN4 Mutant Viruses
Restricted in Replication in Mosquitoes
Section 1. Identification of Viruses Showing Restriction of
Replication in Mosquitoes.
In Examples 1 and 4, DEN4 mutant viruses were generated through
5-FU mutagenesis and charge-cluster-to-alanine mutagenesis,
respectively, in order to identify mutations that confer ts, sp and
att phenotypes. Another highly desirable phenotype of a dengue
virus vaccine is restricted growth in the mosquito host. A dengue
virus vaccine candidate should not be transmissible from humans to
mosquitoes in order to prevent both the introduction of a dengue
virus into an environment in which it is currently not endemic and
to prevent the possible loss of the attenuation phenotype during
prolonged replication in an individual mosquito host. Loss of the
attenuation phenotype could also occur following sustained
transmission between humans and mosquitoes. Recently, loss of
attenuation of a live attenuated poliovirus vaccine was seen
following sustained transmission among humans (CDC 2000 MMWR
49:1094).
In the present example, a panel of 1248 DEN4 mutant viruses
generated through 5-FU mutagenesis and 32 DEN4 mutant viruses
generated through charge-cluster-to-alanine mutagenesis were
assayed for restricted growth in mosquito cells. This is a useful
preliminary assay for restriction in vivo, since restriction in
cultured mosquito cells is often, though not always, associated
with poor infectivity for mosquitoes (Huang, C. Y et al. 2000 J
Virol 74:3020-8). Mutant viruses that showed restriction in
mosquito cells and robust growth in Vero cells (the substrate for
vaccine development, as discussed in Example 6) were targeted for
further characterization.
Generation and characterization of the 5-1A1 mutant. The generation
and isolation of the panel of 1248 5-FU mutant viruses and the
panel of 32 charge-cluster-to-alanine mutant viruses have been
described in Examples 1, 2, and 4. Vero and C6/36 cells were
maintained as described in Example 1.
Each of the 1248 5-FU mutant viruses and 32
charge-cluster-to-alanine mutant viruses was titered in C6/36 cell
monolayers in 24-well plates at 32.degree. C. and 5% CO.sub.2.
After 5 days, plaques were immunostained with anti-DEN4 rabbit
polyclonal antibody and counted as described in the preceding
Examples. Mutant viruses were assayed for one of two phenotypes
indicating restricted growth in mosquito cells: either sp in C6/36
cells relative to Vero cells or a .gtoreq.3.5 log.sub.10PFU/ml
decrease in titer between Vero and C6/36 cells at the permissive
temperature for each cell type. Two mutant viruses, one generated
by 5-FU mutagenesis (#5) and one generated by
charge-cluster-to-alanine mutagenesis (rDEN4-356,357), showed
reduced plaque size in C6/36 cells. After three terminal dilutions,
the 5-FU mutant #5, designated 5-1A1, maintained the reduced plaque
size phenotype. Additionally, recombinant virus rDEN4-7546, tested
for Vero cell adaptation (discussed in detail in Example 6) also
showed reduced plaque size in C6/36 (FIG. 10).
The multicycle growth kinetics of both 5-1A1 and the recombinant
wild type rDEN4 in C6/36 cells were determined as described in
Example 1. Briefly, cells were infected in triplicate at a
multiplicity of infection of 0.01 and samples were harvested at
24-hr intervals. Samples were flash frozen and titered in a single
assay in Vero cell monolayers.
Oral infection of mosquitoes. Aedes aegypti is one of the primary
vectors of dengue virus (Gubler, D. J. 1998 Clin Microbiol Rev
11:480-96). This species was reared at 26.degree. C. and 80%
relative humidity (RH) with a 16 hr daylight cycle. Adults were
allowed continuous access to a cotton pad soaked in a 10% sucrose
solution. Five to ten day old female Ae. aegypti which had been
deprived of a sugar source for 48 hr were fed a bloodmeal
consisting of equal volumes of washed human red blood cells, 10%
sucrose solution, and dengue virus suspension. The infected blood
meal was prepared immediately prior to feeding and offered to
mosquitoes in a water-jacketed feeder covered in stretched parafilm
and preheated to 38.degree. C. (Rutledge, L. C. et al. 1964
Mosquito News 24:407-419). Mosquitoes that took a full bloodmeal
within 45 min were transferred to a new container by aspirator and
maintained as described above. After 21 days, mosquitoes were
stored at -20.degree. C. until dissection.
Intrathoracic inoculation of mosquitoes. The large,
non-haematophagous mosquito Toxorhynchites splendens is a sensitive
host for determining the infectivity of dengue virus. This species
was reared at 24.degree. C. and 75% RH with a 12 hr daylight cycle.
Larvae and pupae were fed on appropriately sized Aedes larvae;
adults were allowed continuous access to a cotton pad soaked in a
10% sucrose solution. Groups of one to ten day old adult T.
splendens of both sexes were immobilized by immersion of their
container in an icewater bath and inoculated intrathoracically with
undiluted virus and serial tenfold dilutions of virus in
1.times.PBS. Virus was inoculated in a 0.22 .mu.l dose using a
Harvard Apparatus microinjector (Medical Systems Corp, Greenvale
N.Y.) and a calibrated glass needle (technique is a modification of
the method described in Rosen and Gubler, 1974).
Detection of viral antigen in body and head tissues by
immunofluorescence assay (IFA). Head and midgut preparations of
Aedes aegypti and head preparations of Toxorhynchites splendens
were made on glass slides as described in Sumanochitrapon et al.
(Sumanochitrapon, W. et al. 1998 Am J Trop Med Hyg 58:283-6).
Slides were fixed in acetone for 20 min, and placed at 4.degree. C.
until processed by IFA. The primary antibody, hyperimmune mouse
ascites fluid specific for DEN-4 (HMAF), was diluted 1/100 in
PBS-Tween 20 (0.05%). Slides were incubated at 37.degree. C. in a
humid chamber for 30 min, and subsequently rinsed in PBS-Tween 20.
The secondary antibody, FITC conjugated goat anti-mouse IgG (KPL,
Gaithersburg, Md.), was diluted 1/200 in PBS-Tween 20 with 0.002%
Evan's Blue. Slides were viewed on an Olympus BX60 microscope. The
infectious dose required to infect 50% of mosquitoes (ID.sub.50)
was determined by the method of Reed and Muench (Reed, L. J. &
Muench, H. 1938 Am J Hyg 27:493-497). For Aedes aegypti infections,
two OID.sub.50 (oral infectious dose 50) values were calculated for
each virus: the OID.sub.50 required to produce an infection in the
midgut, with or without dissemination to the head, and the
OID.sub.50 required to produce disseminated infection. For Tx.
splendens one MID.sub.50 (mosquito infectious dose 50) value was
calculated.
Statistical Analysis. The percentage of mosquitoes infected by
different viruses were compared using logistic regression analysis
(Statview, Abacus Inc.).
Mutations restricting growth of DEN4 in mosquito cells but not Vero
cells are rare. Out of 1280 mutant viruses initially assayed, only
two, #5 and rDEN4-356,357, showed reduced plaque size in C6/36
cells and normal plaque size in Vero cells. One additional virus,
rDEN4-7546 (described in Example 6), with reduced plaque size in
C6/36 was detected in subsequent assays. Mutant virus #5 was cloned
by three successive terminal dilutions and designated 5-1A1;
rDEN4-7546 and rDEN4-356,357 had already been twice-terminally
diluted when they were tested in C6/36 cells. Virus 5-1A1 has been
extensively characterized and its phenotypes are described in
detail in the following section. rDEN4-356,357 and rDEN4-7546 are
envisioned as being characterized in a similar fashion.
Plaque size and growth kinetics of 5-1A1. 5-1A1 replicated to 6.7
log.sub.10PFU/ml in Vero cells with normal plaque size and
replicated to 7.6 log.sub.10PFU/ml in C6/36 cells with small plaque
size (FIG. 6, Table 25). In comparison, wild type DEN4 used as a
concurrent control replicated to 7.3 log.sub.10PFU/ml in Vero
cells, 8.3 log.sub.10PFU/ml in C6/36 cells, and showed normal
plaque size in both cell types (FIG. 6, Table 25). The growth
kinetics of 5-1A1 was compared to that of wild type DEN4 by
infecting C6/36 cells at an MOI of 0.01 and monitoring the
production of infectious virus. The kinetics and magnitude of
replication of 5-1A1 in C6/36 cells was comparable to that of wild
type DEN4 (FIG. 7).
5-1A1 is restricted in its ability to infect mosquitoes. 5-1A1 was
evaluated for its ability to infect Aedes aegypti mosquitoes
through an artificial bloodmeal (Table 26). In this assay the
ability to infect the midgut of the mosquito and the ability for a
midgut infection to disseminate to the head are measured
separately. The oral infectious dose 50 (OID.sub.50) of wild type
DEN4 for the midgut was 3.3 log.sub.10PFU; the OID.sub.50 of wild
type DEN4 for a disseminated infection was 3.9 log.sub.10PFU. In
contrast, 5-1A1 never infected 50% of mosquitoes at the doses used.
In order to calculate the OID.sub.50 for midgut infections by
5-1A1, it was assumed that at a 10-fold higher dose, 100% of 25
mosquitoes would have become infected. Using this assumption, the
conservative estimate of the OID.sub.50 for midgut infections by
5-1A1 was .gtoreq.3.9 log.sub.10PFU. Because 5-1A1 produced only 3
disseminated infections, we did not attempt to calculate an
OID.sub.50 for this category. 5-1A1 was significantly restricted in
its ability to infect the midgut relative to wild type DEN4
(logistic regression, N=150, P<0.001). Additionally, 5-1A1
produced very few disseminated infections, but because of low
numbers this result was not amenable to statistical analysis.
5-1A1 was also significantly restricted in its ability to infect
Tx. splendens mosquitoes following intrathoracic inoculation (Table
27). The MID.sub.50 of wild type DEN4 was 2.3 log.sub.10PFU whereas
the MID.sub.50 of 5-1A1 was estimated to be >3.0 log.sub.10 PFU
(logistic regression, N=36, P<0.01).
5-1A1 does not show a ts or an att phenotype. 5-1A1 was tested for
temperature sensitivity in Vero and HuH-7 cells and for attenuation
in suckling mouse brains as described in Example 1. The mutant
virus was not temperature sensitive, as defined in Example 1, and
was not attenuated in suckling mouse brain (Table 25).
Identification and confirmation of the mutation responsible for the
phenotype of 5-1A1. The nucleotide sequence of the entire genome of
5-1A1 was determined as described in Example 1. Sequencing of 5-1A1
revealed three changes from the wild type sequence: two
translationally-silent point mutations at positions 7359 and 9047,
and one coding point mutation (C to U) at position 7129 in the NS4B
gene which resulted in a proline to leucine substitution.
To formally confirm the effect of the C7129U mutation, the mutation
was inserted into the cDNA p4, which has been described in Examples
1, 2, and 3 and in FIG. 4, using Kunkel mutagenesis as described in
Examples 1 and 3. The mutagenized cDNA was transcribed and
transfected as described in Example 3, and the resulting virus,
after two terminal dilutions, was designated rDEN4-7129-1A. Like
5-1A1, rDEN4-7129-1A showed normal plaque size and titer in Vero
cells and reduced plaque size and normal titer in C6/36 cells
(Table 25). rDEN4-7129-1A was not ts on either Vero or HuH-7 cells
and was not att in suckling mouse brain. Additionally,
rDEN4-7129-1A did not show the SCID-HuH-7 att phenotype described
in Example 3 (Table 25). The ability of rDEN4-7129-1A to infect
mosquitoes is envisioned as being tested in both Ae. aegypti and
Tx. splendens.
To test the compatibility of the C7129U mutation and the .DELTA.30
deletion, the C7129U mutation was inserted into rDEN4.DELTA.30
using previously described techniques. The resulting virus,
designated rDEN4.DELTA.30-7129, is envisioned as being tested for
the phenotypes listed in Table 25.
In summary, three mutant viruses, 5-1A1, rDEN4-356,357 and
rDEN4-7546, showed a particular combination of phenotypes
characterized by normal plaque size and replication to high titers
in Vero cells and small plaque size but unrestricted growth in
mosquito cells. 5-1A1 was further characterized and lacked
temperature sensitivity in either Vero or HuH-7 cells and showed
normal levels of replication in mouse brain and in SCID-HuH-7 mice
and restricted infectivity for both Ae. aegypti and Tx. splendens
mosquitoes. In comparison to wild type rDEN4, the 5-1A1 mutant had
one coding mutation: a point mutation (C to U) at nucleotide 7129
in NS4B resulting in a replacement of Pro with Leu. Because 5-1A1
contains only a single missense mutation, the phenotype of this
mutant virus can be attributed to the effect of the mutation at
position 7129. These results indicate that the 7129 mutation is
responsible for the phenotype of decreased infectivity for
mosquitoes and is predicted to be useful to restrict replication of
vaccine candidates in mosquitoes. To formally confirm this, we have
inserted the 7129 mutation into a recombinant DEN4 virus. The
resulting virus, designated rDEN4-7129-1A, shows an absence of ts
and att phenotypes similar to 5-1A1. It is envisioned as being
tested for mosquito infectivity.
The 7129 mutation is a valuable point mutation to include in a DEN4
vaccine candidate and into each of the dengue virus antigenic
chimeric vaccine candidates since its biological activity is host
specific, i.e., it is restricted in replication in mosquitoes but
not in mammals. Moreover, as discussed in Example 6, the 7129
mutation has also been shown to enhance replication in Vero cells.
Thus, its insertion into a vaccine candidate is envisioned as
enhancing vaccine production in tissue culture without affecting
the biological properties specified by other attenuating mutations.
It is also envisioned as providing a useful safeguard against
mosquito transmission of a dengue virus vaccine.
Section II. Design of Mutations to Restrict Replication in
Mosquitoes
In Section 1 of Example 5, we screened a large panel of mutant
viruses carrying both random mutations (generated with
5-fluorouracil) and specific mutations (generated through
charge-cluster-to-alanine mutagenesis) for restricted growth in
C6/36 cells, a proxy measure for restriction in mosquitoes.
However, in neither case were mutations designed for the specific
purpose of restricting replication in mosquitoes. In this section,
we identified nucleotide sequences in the 3' UTR that show
conserved differences between the mosquito-transmitted and
tick-transmitted flaviviruses. We then altered those sequences in
the DEN4 cDNA p4 by either deleting them altogether or exchanging
them with the homologous sequence of the tick-transmitted Langat
virus. The resulting viruses were assayed for reduced plaque size
and titer in both Vero and C6/36 cells and for infectivity for Ae.
aegypti and Tx. splendens.
Identification and modification of particular 3' UTR sequences
showing conserved differences between vectors. Several studies
(Olsthoom, R. C. & Bol, J. F. 2001 RNA 7:1370-7; Proutski, V.
et al. 1997 Nucleic Acids Res 25:1194-202) have identified
conserved differences in the nucleotide sequences of the 3' UTR of
mosquito-transmitted and tick-transmitted flaviviruses. Such
differences are concentrated in the 3' terminal core region, the
approximately 400 3' terminal nucleotides. It has been suggested
that these sequences may have a vector-specific function (Proutski,
V. et al. 1997 Nucleic Acids Res 25:1194-202). While such a
function has not been identified, it may nonetheless be possible to
disrupt vector infectivity by deleting or otherwise altering these
nucleotides.
To identify target sequences for this type of alteration, we
constructed an alignment of the 3' UTR nucleotide sequences of
seven mosquito-transmitted flaviviruses and four tick-transmitted
flaviviruses (FIG. 8). From this alignment, we identified several
sequences that showed conserved differences between the
mosquito-transmitted flaviviruses and tick-transmitted
flaviviruses. We then designed primers to alter these sequences in
the wt DEN4 cDNA p4 (FIG. 4) in one of two ways: 1) deletion of the
nucleotides (.DELTA.) or 2) replacement of the nucleotides with the
homologous sequence from the tick-transmitted flavivirus Langat
(swap). Langat was chosen as the template for swapped nucleotides
because it is naturally attenuated (Pletnev, A. G. 2001 Virology
282:288-300), and therefore unlikely to enhance the virulence of
rDEN4 virus derived from the modified cDNA. The DEN4 sequences
altered and the mutagenesis primers used to do so are listed in
Table 28. Nucleotides 10508-10530 correspond to the CS2 region
identified in previous studies (Proutski, V. et al. 1997 Nucleic
Acids Res 25:1194-202).
Mutagenesis of p4, transcription and transfection were conducted as
previously described in Section I of this Example. All five of the
engineered viruses were recovered, and all were subjected to two
rounds of terminal dilution as previously described.
Evaluation of phenotypes: cell culture. Viruses were titered in
Vero and C6/36 cells as previously described, and the results are
listed in Table 29. All of the viruses replicated to >5.0
log.sub.10PFU/ml; one of them (rDEN4.DELTA.10508-10530) replicated
to >8.0 log.sub.10PFU/ml. Only one of the viruses
(rDEN4.DELTA.10535-10544) was small plaque in C6/36 cells; this
virus showed wild-type plaque size in Vero cells. Interestingly,
another virus (rDEN4swap10508-10539) showed wild type plaque size
in C6/36 cells but was sp in Vero cells.
Evaluation of phenotypes: mosquito infectivity. To date one of the
five viruses has been tested for infectivity via intrathoracic
inoculation in Tx. splendens, using previously described methods.
Virus rDEN4.DELTA.10508-10530 was dramatically restricted in
infectivity relative to the wild type (Table 30). So few mosquitoes
were infected that it was not possible to calculate an MID.sub.50
for this virus.
One of the five viruses has been tested for infectivity of Ae.
aegypti fed on an infectious bloodmeal using previously described
methods. rDEN4swap10535-10544 (Table 31) caused significantly fewer
midgut infections than wild type rDEN4, but the percentage of
disseminated infections did not differ between rDEN4swap10535-10544
and wild type rDEN4. All of the viruses are envisioned as being
tested for mosquito infectivity using both methods.
Summary. In this example we have outlined two different strategies
for preventing mosquito transmission of a dengue vaccine. First,
several small substitution mutations, including two point mutations
and one paired charge-to-alanine substitution, have been shown to
restrict the replication of DEN4 in mosquito C6/36 cells in cell
culture, and one of these mutations (C7129U) has been shown to
restrict the ability of DEN4 virus to infect mosquitoes. Second, we
have created a variety of deletion and substitution mutations in
regions of the DEN4 3' UTR that show conserved differences between
mosquito-transmitted and tick-transmitted flaviviruses. One of
these viruses is sp in C6/36 cells and at least two of these
viruses show some degree of restriction of mosquito infectivity. By
design, the nucleotide sequences in which these mutations were made
are highly conserved among the four dengue serotypes and among
mosquito-transmitted flaviviruses in general, indicating that they
are portable to other vaccine candidates for mosquito-borne
flaviviruses. All of the mutations discussed in this Example lie
outside the structural genes and so are envisioned as being useful
in constructing antigenic-chimeric vaccine candidates.
Example 6
Adaptation Mutations which Enhance the Replication of DEN4 and DEN4
Chimeric Viruses in Vero Cells
Vero cells are a highly characterized substrate that should be
suitable for the manufacture of live attenuated flavivirus
vaccines, such as dengue virus and tick-borne encephalitis virus.
In addition, Vero cells can also be used to grow flaviviruses to
high titer for the preparation of an inactivated virus vaccine.
Optimal sequences for the efficient growth of dengue viruses in
Vero cells have not been identified, but it is well known that
flaviviruses accumulate mutations during passage in various cell
cultures (Dunster, L. M. et al. 1999 Virology 261:309-18; Theiler,
M. & Smith, H. H. 1937 J Exp Med 65:787-800). Inclusion of
specific sequences in live attenuated viruses that enhance their
replication in Vero cells and increase the number of doses of
vaccine produced per unit substrate would greatly facilitate their
manufacture. Similarly, inclusion of Vero cell growth-promoting
sequences in wild type viruses used for the preparation of an
inactivated virus vaccine would also greatly facilitate the
manufacture of the vaccine. The present example identifies
mutations that occur following passage of DEN4 virus and DEN2/4
chimeric viruses in Vero cells. Data derived from five sources
provided information for this analysis making it possible to
generate a list of Vero cell growth-promoting sequences.
Presence of identical mutations in multiple 5-FU mutant viruses.
First, as described in Examples 1 and 2, the genomes of 42 dengue
virus clones isolated from a 5-FU mutagenized stock of virus were
completely sequenced. If mutations that enhance replication
occurred during the passage of these 42 mutant viruses in Vero
cells, then such mutations should reveal themselves by
representation in more than one clone. Analysis of the 42 sequences
revealed the occurrence of specific missense mutations in coding
regions or nucleotide substitutions in UTRs in multiple clones that
are not present in the 2A parental virus genome (Tables 11 and 32).
These mutations, many of which occur within a 400 nucleotide
section of the NS4B coding region, represent Vero cell-adaptation
mutations. One mutation, such as the 4995 mutation, present in
eight viruses was found to specify both ts and att phenotypes
(Examples 1 and 3). In contrast, the 7163 mutation, present in six
viruses, does not specify a ts or att phenotype (Table 13) and thus
is an example of a specific Vero cell growth-promoting
mutation.
Presence of Vero cell adaptation mutations in other DEN4 viruses
and DEN2/4 antigenic chimeric viruses. Second, the 2A-13 dengue
virus that was used as a parallel passaged wild type control during
the 5-FU experiments described in Example 1 was grown and cloned in
Vero cells in the absence of 5-FU in a manner identical to that of
the 5-FU treated viruses. Sequence analysis of this 5-FU untreated
virus, designated 2A-13-1A1, revealed that the virus genome
contained a mutation at nucleotide 7163 (Example 1 and Table 32),
identical to the missense mutation previously identified in 6 of
the 5-FU mutant viruses (Tables 11 and 32). This indicates that
growth and passage of DEN4 virus in Vero cells is sufficient to
acquire this specific mutation, i.e. mutagenesis with 5-FU is not
required. Thus, information from two separate sources indicates
that the 7163 mutation appeared in separate Vero cell passaged
viruses, thereby strengthening the interpretation that this
mutation is growth promoting.
Third, following passage of the 2A.DELTA.30 and rDEN4.DELTA.30 in
Vero cells, sequence analysis revealed the presence of a mutation
at nucleotides 7153 and 7163, respectively. These two mutations
were also previously identified among the 5-FU treated viruses
(Table 32). Again, identical mutations appeared following
independent passage of virus in Vero cells, corroborating the
hypothesis that these mutations confer a growth advantage in Vero
cells.
Fourth, an antigenic chimeric dengue virus vaccine candidate was
generated that expressed the structural proteins C, prM, and E from
DEN2 on a DEN4 wild type genetic background or an attenuated
.DELTA.30 genetic background. To construct this virus, the C, prM
and E region of wild type cDNA plasmid p4 was replaced with a
similar region from DEN2 virus strain NGC (FIG. 10). Specifically,
nucleotides between restriction sites Bg/II (nt 88) and XhoI (nt
2345) of p4 were replaced with those derived from dengue type 2
virus. RNA transcripts synthesized from the resulting p4-D2 plasmid
were transfected into Vero cells and rDEN2/4 virus was recovered. A
further attenuated version of this chimeric virus containing the
.DELTA.30 mutation, rDEN2/4.DELTA.30, was recovered in C6/36
mosquito cells following transfection of cells with RNA transcripts
derived p4.DELTA.30-D2. However, rDEN2/4.DELTA.30 could not be
recovered directly in Vero cells. The rDEN2/4.DELTA.30 mutant virus
recovered in C6/36 cells replicated to very low levels in Vero
cells (<1.0 log.sub.10PFU/ml) but grew to high titer in C6/36
cells (>6.0 log.sub.10PFU/ml). Genomic sequence of the
C6/36-derived virus matched the predicted cDNA sequence and is
shown in Appendix 3. Nevertheless, when C6/36-derived
rDEN2/4.DELTA.30 was serially passaged 3 to 4 times in Vero cells,
a virus population adapted for growth in Vero cells emerged. Virus
from this Vero cell-adapted preparation was cloned and amplified in
Vero cells to a titer >6.0 log.sub.10PFU/ml. The genomic
sequence was determined for 2 independent virus clones and compared
to the predicted cDNA sequence (Table 33 and 34). Each cloned virus
contains a mutation in a non-structural gene which coincides
closely in location or sequence with a mutation previously
identified among the panel of 5-FU mutagenized viruses. The other
mutations in these two clones also might confer a growth advantage
in Vero cells. Importantly, the mutations identified in Tables 33
and 34 are absolutely required for replication in Vero cells, and
it would not be possible to produce the rDEN2/4.DELTA.30 vaccine
candidate in Vero cells without the growth-promoting mutations
identified in Tables 33 and 34.
Fifth, sequence analysis of the dengue 4 wild-type virus strain
814669 (GenBank accession no. AF326573) following passage in Vero
cells identified a mutation in the NS5 region at nucleotide 7630
which had previously been identified among the panel of 5-FU
mutagenized viruses (Table 32). This mutation at nucleotide 7630
was introduced into recombinant virus rDEN4 by site-directed
mutagenesis as described in Table 16. The resulting virus,
rDEN4-7630, was not temperature sensitive when tested at 39.degree.
C., indicating that mutation 7630 does not contribute to
temperature sensitivity.
Characterization of rDEN2/4.DELTA.30 chimeric viruses containing
single and multiple Vero cell adaptation mutations. The generation
of chimeric virus rDEN2/4.DELTA.30 provided a unique opportunity
for evaluating the capacity of individual mutations to promote
increased growth in Vero cells. Because rDEN2/4.DELTA.30 replicates
to very low titer in Vero cells, yet can be efficiently generated
in C6/36 mosquito cells, recombinant virus bearing putative
Vero-cell adapting mutations were first generated in C6/36 cells
and then virus titers were determined in both C6/36 and Vero cells.
As shown in Table 35, addition of a single mutation to
rDEN2/4.DELTA.30 resulted in a greater than 1000-fold increase in
titer in Vero cells, confirming the Vero cell adaptation phenotype
conferred by these mutations. However, the combination of two
separate mutations into a single virus did not increase the titer
in Vero cells beyond the level observed for viruses bearing a
single adaptation mutation. Inclusion of either the 7182 or 7630
mutation in the cDNA of rDEN2/4.DELTA.30 allowed the virus to be
recovered directly in Vero cells, circumventing the need to recover
the virus in C6/36 cells.
Characterization of the growth properties of rDEN4 viruses
containing single and multiple defined Vero cell adaptation
mutations. To confirm the ability of Vero cell adaptation mutations
to enhance growth of DEN4 viruses, site-directed mutagenesis was
used to generate rDEN4 viruses encoding selected individual
mutations as described in Examples 1 and 3. Five mutations in NS4B
(7153, 7162, 7163, 7182, and 7546) from the list of repeated
mutations in the 5-FU mutant viruses (Table 32) were introduced
singly into the p4 cDNA clone. In addition, the
mosquito-restricted, rDEN4-7129 virus was evaluated for enhanced
growth in Vero cells since the location of this mutation is in the
same region of NS4B. Each virus, including wild-type rDEN4, was
recovered, terminally diluted, and propagated in C6/36 cells to
prevent introduction of additional Vero cell adaptation mutations,
however, because of its restricted growth in C6/36 cells,
rDEN4-7129 was propagated only in Vero cells.
Plaque size was evaluated for each mutant rDEN4 virus in Vero cells
and C6/36 cells and compared to wild-type rDEN4. Six-well plates of
each cell were inoculated with dilutions of virus and plaques were
visualized five days later. Representative plaques are illustrated
in FIG. 10 and demonstrate that the presence of a Vero cell
adaptation mutation does indeed confer increased virus cell to cell
spread and growth specifically in Vero cells. In C6/36 cells,
average plaque size was approximately 0.50 mm for both wild-type
rDEN4 and each mutant virus (except for rDEN4-7546 and rDEN4-7129
which were smaller than wild-type; see Example 5). However, rDEN4
viruses expressing mutation 7162, 7163, 7182, and 7129 had a
greater than two-fold increase in plaque size in Vero cells
compared to wild-type rDEN4 virus. A smaller but consistent
increase in plaque size was observed for rDEN4-7153 and
rDEN4-7546.
Growth kinetics and virus yield in Vero cells was assessed for the
same panel of rDEN4 viruses. Vero cells were infected at an MOI of
0.01 and samples were removed daily for 10 days, titered on Vero
cells, and plaques were visualized. The results in FIG. 11 indicate
that the presence of a Vero cell adaptation mutation increased the
kinetics of virus growth, but had only a marginal effect on the
peak virus yield. At day four post-infection, wild-type rDEN4 grew
to 5.2 log.sub.10PFU/ml while the level of replication in
rDEN4-7129-infected cells was 100-fold higher. The rest of the
mutant rDEN4 viruses had an increased yield at day four ranging
from 0.9 (rDEN4-7153) to 1.6 (rDEN4-7162 and -7163)
log.sub.10PFU/ml. Interestingly, enhanced kinetics of virus growth
correlated with increased plaque size in Vero cells. The peak virus
yield was reached by day 6 post-infection for rDEN4-7129, -7162,
-7163, and -7182 while wild-type rDEN4 did not reach peak titer
until day 10. However, the peak virus yield was only slightly
higher in rDEN4 viruses expressing Vero cell adaptation
mutations.
In an effort to further enhance rDEN4 replication, especially the
peak virus yield, combinations of selected Vero cell adaptation
mutations were introduced into the rDEN4 background. Three viruses
with dual mutations were generated: rDEN4-7153-7163,
rDEN4-7153-7182, and rDEN4-7546-7630 and tested in a Vero cell time
course infection as described above along with rDEN4 and rDEN4-7162
as a positive control (FIG. 12). The viruses expressing combined
mutations grew in a nearly identical manner to rDEN4-7162
indicating that these selected combinations did not enhance the
kinetics or peak virus yield. Additional combinations of these and
other Vero cell adaptation mutations are envisioned as increasing
peak virus yield.
Discussion. Some of the growth promoting mutations listed in Table
32 are also found in homologous regions of DEN1, DEN2, and DEN3 and
are envisioned as serving to promote the replication of these
viruses in Vero cells. Specifically, the growth promoting mutations
indicated in Table 32 that are present in a DEN4 virus are
envisioned as being useful for importation into homologous regions
of other flaviviruses, such as DEN1, DEN2 and DEN3. Examples of
such conserved regions are shown in Appendix 4 and are listed in
Table 36. The nucleotides for both mutation 7129 and 7182 are
conserved in all four dengue virus serotypes. It is also
interesting to note that mutation 7129 not only increases growth in
Vero cells (FIG. 10), but it also forms small plaques in mosquito
cells (FIG. 6, Table 25). Lee et al. previously passaged DEN3 virus
in Vero cells and performed limited sequence analysis of only the
structural gene regions of the resulting viruses (Lee, E. et al.
1997 Virology 232:281-90). From this analysis a menu of Vero
adaptation mutations was assembled. Although none of these
mutations correspond to the Vero adaptation mutations identified in
this Example, a single mutation at amino acid position 202 in DEN3
corresponds to mutation 1542 identified in 5-FU mutant virus #1012.
The current Example emphasizes the importance in this type of study
of determining the sequence of the entire viral genome.
Vero cell growth optimized viruses are envisioned as having
usefulness in the following areas. First, the yield of a live
attenuated vaccine virus in Vero cells is predicted to be
augmented. The live attenuated vaccine candidate is conveniently a
DEN4 or other dengue virus or a DEN 1/4, DEN2/4, or DEN3/4
antigenic chimeric virus, or a chimeric virus of another flavivirus
based on the DEN4 background. The increased yield of vaccine virus
is envisioned as decreasing the cost of vaccine manufacture.
Second, Vero cell adaptation mutations that are attenuating
mutations, such as the 4995 mutation, are envisioned as being
stable during the multiple passage and amplification of virus in
Vero cell cultures that is required for production of a large
number of vaccine doses. Third, Vero cell adaptation mutations are
actually required for the growth of the rDEN2/4.DELTA.30 vaccine
candidate in Vero cells. Fourth, the increase in yield of a DEN
wild type or an attenuated virus is envisioned as making it
economically feasible to manufacture an inactivated virus vaccine.
Fifth, the presence of the Vero cell growth promoting mutations in
the DEN4 vector of the rDEN1/4, rDEN2/4, and rDEN3/4 antigenic
chimeric viruses or other flavivirus chimeric viruses based on DEN4
is envisioned as permitting the viruses to grow to a high titer and
as thereby being useful in the manufacture of a inactivated virus
vaccine. Sixth, the insertion of Vero cell growth promoting
mutations into cDNAs such as rDEN2/4.DELTA.30 is envisioned as
permitting recovery of virus directly in Vero cells, for which
there are qualified master cell banks for manufacture, rather than
in C6/36 cells for which qualified cell banks are not available.
And seventh, insertion of the 7129 and 7182 mutations into DEN1,
DEN2, or DEN3 wt viruses is envisioned as increasing their ability
to replicate efficiently and be recovered from cDNA in Vero
cells.
Example 7
Assembly of a List of Attenuating Mutations
The data presented in these examples permits the assembly of a list
of attenuating mutations that is summarized in Table 37. This list
contains individual mutations identified in Tables 13-16, 20, and
21 that are known to independently specify an attenuation
phenotype. Mutation 7129 is also included since it is derived from
virus 5-1A1 shown to be attenuated in mosquitoes. We envision using
various combinations of mutations from this list to generate
viruses with sets of desirable properties such as restricted growth
in the liver or in the brain as taught in Example 3 (Table 18) and
Example 4 (Tables 23 and 24). These mutations are also combinable
with other previously described attenuating mutations such as the
.DELTA.30 mutation, as taught in Example 1 (Table 6) and Example 3
(Table 19) to produce recombinant viruses that are satisfactorily
attenuated and immunogenic. Mutations listed in Table 37 are also
envisioned as being combined with other previously described
attenuating mutations such as other deletion mutations or other
point mutations (Blok, J. et al. 1992 Virology 187:573-90;
Butrapet, S. et al. 2000 J Virol 74:3011-9; Men, R. et al. 1996
Virol 70:3930-7; Puri, B. et al. 1997 J Gen Virol 78:2287-91).
The possibility of importing an attenuating mutation present in one
paramyxovirus into a homologous region of a second paramyxovirus
has recently been described (Durbin, A. P. et al. 1999 Virology
261:319-30; Skiadopoulos, M. H. et al. 1999 Virology 260:125-35).
Such an importation confers an att phenotype to the second virus
or, alternatively, further attenuates the virus for growth in vivo.
Similarly we envision importing an attenuating mutation present in
one flavivirus to a homologous region of a second flavivirus which
would confer an att phenotype to the second flavivirus or,
alternatively, would further attenuate the virus for growth in
vivo. Specifically, the attenuating mutations indicated in Table 37
are envisioned as being useful for importation into homologous
regions of other flaviviruses. Examples of such homologous regions
are indicated in Appendix 4 for the mutations listed in Table
37.
Example 8
Evaluation of Dengue Virus Vaccine in Humans and Rhesus Monkeys
The present example evaluates the attenuation for humans and rhesus
monkeys (as an animal model) of a DEN-4 mutant bearing a 30
nucleotide deletion (.DELTA.30) that was introduced into its 3'
untranslated region by site-directed mutagenesis and that was found
previously to be attenuated for rhesus monkeys (Men, R. et al. 1996
J Virol 70:3930-7), as representative of the evaluation of any
dengue virus vaccine for attenuation in humans and rhesus monkeys
(as an animal model).
Viruses and cells. The wild type (wt) DEN-4 virus strain 814669
(Dominica, 1981), originally isolated in Aedes pseudoscutellaris
(AP61) cells, was previously plaque-purified in LLC-MK2 cells and
amplified in C6/36 cells as described (Mackow, E. et al. 1987
Virology 159:217-28). For further amplification, the C6/36
suspension was passaged 2 times in Vero (WHO) cells maintained in
MEM-E (Life Technologies, Grand Island, N.Y.) supplemented with 10%
FBS. Viruses derived from RNA transfection or used for clinical lot
development were grown in Vero (WHO) cells maintained in serum-free
media, VP-SFM (Life Technologies).
Construction of DEN-4 deletion mutants. A 30 nucleotide (nt)
deletion was previously introduced into the 3' untranslated region
of the 2A cDNA clone of wt DEN-4 strain 814669 as described (Men,
R. et al. 1996 J Virol 70:3930-7). This deletion removes
nucleotides 10478-10507, and was originally designated 3'd 172-143,
signifying the location of the deletion relative to the 3' end of
the viral genome. In the current example, this deletion is referred
to as .DELTA.30. The full-length 2A cDNA clone has undergone
several subsequent modifications to improve its ability to be
genetically manipulated. As previously described, a
translationally-silent XhoI restriction enzyme site was engineered
near the end of the E region at nucleotide 2348 to create clone
2A-XhoI (Bray, M. & Lai, C. J. 1991 PNAS USA 88:10342-6). In
this example, the viral coding sequence of the 2A-XhoI cDNA clone
was further modified using site-directed mutagenesis to create
clone p4: a unique BbvCI restriction site was introduced near the
C-prM junction (nucleotides 447-452); an extra XbaI restriction
site was ablated by mutation of nucleotide 7730; and a unique SacII
restriction site was created in the NS5 region (nucleotides
9318-9320). Each of these engineered mutations is translationally
silent and does not change the amino acid sequence of the viral
polypeptide. Also, several mutations were made in the vector region
of clone p4 to introduce or ablate additional restriction sites.
The cDNA clone p4.DELTA.30 was generated by introducing the
.DELTA.30 mutation into clone p4. This was accomplished by
replacing the MluI-KpnI fragment of p4 (nucleotides 10403-10654)
with that derived from plasmid 2A.DELTA.30 containing the 30
nucleotide deletion. The cDNA clones p4 and p4.DELTA.30 were
subsequently used to generate recombinant viruses rDEN4 and
rDEN4.DELTA.30, respectively.
Generation of viruses. Full-length RNA transcripts were synthesized
from cDNA clones 2A and 2A.DELTA.30 using SP6 RNA polymerase as
previously described (Lai, C. J. et al. 1991 PNAS USA 88:5139-43;
Men, R. et al. 1996 J Virol 70:3930-7). The reaction to generate
full-length RNA transcripts from cDNA clones p4 and p4.DELTA.30 was
modified and consisted of a 50 .mu.l reaction mixture containing 1
.mu.g linearized plasmid, 60 U SP6 polymerase (New England Biolabs
(NEB), Beverly, Mass.), 1.times.RNA polymerase buffer (40 mM
Tris-HCl, pH 7.9, 6 mM MgCl.sub.2, 2 mM spermidine, 10 mM
dithiothreitol), 0.5 mM m7G(5')ppp(5')G cap analog (NEB), 1 mM each
nucleotide triphosphate, 1 U pyrophosphatase (NEB), and 80 U RNAse
inhibitor (Roche, Indianapolis, Ind.). This reaction mixture was
incubated at 40.degree. C. for 90 min and the resulting transcripts
were purified using RNeasy mini kit (Qiagen, Valencia, Calif.). For
transfection of Vero cells, purified transcripts (1 .mu.g) were
mixed with 12 .mu.l DOTAP liposome reagent (Roche) in saline
containing 20 mM HEPES buffer (pH 7.6) and added to cell monolayer
cultures in a 6-well plate. After 5-17 days, tissue culture medium
was harvested, clarified by centrifugation, and virus was amplified
in Vero cells. The presence of virus was confirmed by plaque
titration. It should be noted that during the course of
transfection and amplification of 2A.DELTA.30 to create the vaccine
lot, the virus underwent a total of 6 passages entirely in Vero
cells. The remaining viruses, rDEN4 and rDEN4.DELTA.30 were
passaged 5 times in Vero cells to generate the virus suspension
used for sequence analysis and studies in rhesus monkeys.
Vaccine Production. An aliquot of clarified tissue culture fluid
containing vaccine candidate 2A.DELTA.30 was submitted to DynCorp
(Rockville, Md.) for amplification of virus in Vero cells and
production of a vaccine lot. For vaccine production, 2A.DELTA.30
infected tissue culture supernatant was harvested, SPG buffer added
(final concentration: 218 mM sucrose, 6 mM L-glutamic acid, 3.8 mM
potassium phosphate, monobasic, and 7.2 mM potassium phosphate,
dibasic, pH 7.2), and the virus suspension was clarified by low
speed centrifugation. To degrade residual Vero cell DNA, the
vaccine suspension was treated with Benzonase endonuclease
(American International Chemical, Natick, Mass.), 100 U/ml and
incubated for 1 hr at 37.degree. C., followed by high-speed
centrifugation (17,000.times.g, 16 hr). The resulting virus pellet
was gently rinsed with MEM-E, resuspended in MEM-E containing SPG,
sonicated, distributed into heat-sealed ampoules, and stored frozen
at -70.degree. C. Final container safety testing confirmed
microbial sterility, tissue culture purity, and animal safety. The
2A.DELTA.30 vaccine lot (designated DEN4-9) has a titer of 7.48
log.sub.10PFU/ml, with a single dose of 5.0 log.sub.10PFU/ml
containing <1 pg/ml Vero cell DNA and <0.001 U/ml Benzonase
endonuclease.
Sequence of cDNA clones and viral genomes. The nucleotide sequence
of the viral genome region of cDNA plasmids 2A and p4 was
determined on a 310 genetic analyzer (Applied Biosystems, Foster
City, Calif.) using vector-specific and DEN-4-specific primers in
BigDye terminator cycle sequencing reactions (Applied Biosystems).
The nucleotide sequence of the genomes of the parental wt DEN-4
strain 814669 and of recombinant viruses 2A wt, 2A.DELTA.30
(vaccine lot), rDEN4, and rDEN4.DELTA.30 was also determined. Viral
RNA was extracted from virus preparations and serum samples using
the QIAamp Viral RNA mini kit (Qiagen). Reverse transcription (RT)
was performed using random hexamers and the Super-Script
First-Strand Synthesis System for RT-PCR (Life Technologies).
Overlapping PCR fragments of approximately 2000 base pairs were
generated using optimized DEN-4 specific primers and Advantage cDNA
polymerase (ClonTech, Palo Alto, Calif.). Both strands of purified
PCR fragments were sequenced directly using dye-terminator
reactions as described above and results were assembled into a
consensus sequence. To determine the nucleotide sequence of the
viral RNA 5' and 3' regions, the 5' cap nucleoside of the viral RNA
was removed with tobacco acid pyrophosphatase (Epicentre, Madison,
Wis.) followed by circularization of the RNA using RNA ligase
(Epicentre). RT-PCR was performed as described and a cDNA fragment
spanning the ligation junction was sequenced using DEN-4 specific
primers. GenBank accession numbers have been assigned as follows
(virus: accession number): 814669: AF326573, 2A.DELTA.30: AF326826,
rDEN4: AF326825, and rDEN4.DELTA.30: AF326827.
Human Vaccine Recipients. 20 normal healthy adult volunteers were
recruited by the Johns Hopkins School of Hygiene and Public Health
Center for Immunization Research (CIR) located in Baltimore, Md.
The clinical protocol was reviewed and approved by the Joint
Committee for Clinical Investigation of the Johns Hopkins
University School of Medicine and informed consent was obtained
from each volunteer. Volunteers were enrolled in the study if they
met the following inclusion criteria: 18-45 years of age; no
history of chronic illness; a normal physical examination; human
immunodeficiency virus antibody negative, hepatitis B surface
antigen negative, and hepatitis C antibody negative; no stool
occult blood; and normal values for complete blood cell count (CBC)
with differential, hematocrit, platelet count, serum creatinine,
serum aspartate amino transferase (AST), alanine amino transferase
(ALT), alkaline phosphatase, bilirubin, prothrombin time (PT),
partial thromboplastin time (PTT), and urinalysis. Female
volunteers were required to have a negative urine pregnancy test
prior to vaccination and on the day of vaccination and to agree to
use contraception or abstain from sexual intercourse for the
duration of the study. Volunteers also lacked serological evidence
of prior flavivirus infection as defined by
hemagglutination-inhibition antibody titer <1:10 to DEN-1,
DEN-2, DEN-3, DEN-4, St. Louis encephalitis virus, Japanese
encephalitis virus, or yellow fever virus and a plaque-reduction
neutralization titer <1:10 to DEN-4 and yellow fever virus.
Studies in Humans. Volunteers were immunized in three successive
cohorts of four, six, and ten volunteers to assess the safety of
the vaccine. In this study, an illness was defined as the
following: dengue virus infection associated with a platelet count
of <90,000/mm.sup.3; serum ALT >4 times normal; oral
temperature >38.degree. C. for >2 successive days; or
headache and/or myalgia lasting >2 successive days. Systemic
illness was defined as the occurrence of fever >38.degree. C.
for >2 consecutive days, or any 2 of the following for at least
two consecutive study days: headache, malaise, anorexia, and
myalgia/arthralgia. The trials were conducted between October and
April, a time of low mosquito prevalence, to reduce the risk of
transmission of vaccine virus from the volunteers to the
community.
On the day of vaccination, vaccine candidate 2A.DELTA.30 was
diluted to 5.3 log.sub.10PFU/ml in sterile saline for injection,
USP, and each volunteer was injected subcutaneously with a 0.5 ml
containing 5.0 log.sub.10PFU of vaccine into the left deltoid
region. Volunteers were given a home diary card on which they were
to record their temperature twice daily for days 0-5
post-vaccination. The volunteers returned to the clinic each day
for examination by a physician and their diary cards were reviewed.
The injection site was evaluated for erythema, induration, and
tenderness. Clinical signs and symptoms such as headache, rash,
petechiae, lymphadenopathy, hepatomegaly, abdominal tenderness,
anorexia, nausea, fatigue, myalgia, arthralgia, eye pain, and
photophobia were assessed daily. Symptoms were graded as mild (no
need for treatment or a change in activity), moderate (treatment
needed or change in activity noted, yet still able to continue
daily activity) or severe (confined to bed). Blood was drawn for
CBC with differential and for virus quantitation on days 0, 2 and
4. Volunteers were admitted to the inpatient unit at the CIR on the
sixth day after immunization. The study physician evaluated all
volunteers each day by physical examination and interview. The
volunteers had their blood pressure, pulse, and temperature
recorded four times a day. Blood was drawn each day for CBC with
differential and for virus quantitation and every other day for ALT
measurement. Volunteers were confined to the inpatient unit until
discharge on study day 15. On study days 28 and 42, volunteers
returned for physical examination and blood was drawn for virus
quantitation (day 28) and for serum antibody measurement (day 28
and 42).
Virus quantitation and amplification. Serum was obtained for
detection of viremia and titration of virus in positive specimens.
For these purposes 8.5 ml of blood was collected in a serum
separator tube and incubated at room temperature for less than 30
min. Serum was decanted into 0.5 ml aliquots, rapidly frozen in a
dry ice/ethanol bath and stored at -70.degree. C. Serum aliquots
were thawed and serial 10-fold dilutions were inoculated onto Vero
cell monolayer cultures in 24-well plates. After one hour
incubation at room temperature, the monolayers were overlaid with
0.8% methylcellulose in Opti-MEM (Life Technologies) supplemented
with 5% fetal bovine serum (FBS). Following incubation at
37.degree. C. for four days, virus plaques were visualized by
immunoperoxidase staining. Briefly, cell monolayers were fixed in
80% methanol for 30 min and rinsed with antibody buffer (5% nonfat
milk in phosphate buffered saline). Rabbit polyclonal DEN-4
antibodies were diluted 1:1000 in antibody buffer and added to each
well followed by a one hr incubation at 37.degree. C. Primary
antibody was removed and the cell monolayers were washed twice with
antibody buffer. Peroxidase-labelled goat-anti-rabbit IgG (KPL,
Gaithersburg, Md.) was diluted 1:500 in antibody buffer and added
to each well followed by a one hr incubation at 37.degree. C.
Secondary antibody was removed and the wells were washed twice with
phosphate buffered saline. Peroxidase substrate (4 chloro-1-napthol
in H.sub.2O.sub.2) was added to each well and visible plaques were
counted.
For amplification of virus in serum samples, a 0.3 ml aliquot of
serum was inoculated directly onto a single well of a 6-well plate
of Vero cell monolayers and incubated at 37.degree. C. for 7 days.
Cell culture fluid was then assayed for virus by plaque assay as
described above.
Serology. Hemagglutination-inhibition (HAI) assays were performed
as previously described (Clarke, D. H. & Casals, J. 1958 Am J
Trop Med Hyg 7:561-73). Plaque-reduction neutralization titers
(PRNT) were determined by a modification of the technique described
by Russell (Russell, P. K. et al. 1967 J Immunol 99:285-90).
Briefly, test sera were heat inactivated (56.degree. C. for 30 min)
and serial 2-fold dilutions beginning at 1:10 were made in OptiMEM
supplemented with 0.25% human serum albumin. rDEN4.DELTA.30 virus,
diluted to a final concentration of 1000 PFU/ml in the same
diluent, was added to equal volumes of the diluted serum and mixed
well. The virus/serum mixture was incubated at 37.degree. C. for 30
min. Cell culture medium was removed from 90% confluent monolayer
cultures of Vero cells on 24-well plates and 50 .mu.l of
virus/serum mixture was transferred onto duplicate cell monolayers.
Cell monolayers were incubated for 60 min at 37.degree. C. and
overlaid with 0.8% methylcellulose in OptiMEM supplemented with 2%
FBS. Samples were incubated at 37.degree. C. for 4 days after which
plaques were visualized by immunoperoxidase staining as described
above, and a 60% plaque-reduction neutralization titer was
calculated.
Studies in rhesus monkeys. Evaluation of the replication and
immunogenicity of wt virus 814669, and recombinant viruses 2A wt,
2A.DELTA.30 (vaccine lot), rDEN4, and rDEN4.DELTA.30 in juvenile
rhesus monkeys was performed as previously described (Men R. et al.
1996 J Virol 70:3930-7). Briefly, dengue virus seronegative monkeys
were injected subcutaneously with 5.0 log.sub.10PFU of virus
diluted in L-15 medium (Quality Biological, Gaithersburg, Md.)
containing SPG buffer. A dose of 1 ml was divided between two
injections in each side of the upper shoulder area. Monkeys were
observed daily and blood was collected on days 0-10 and 28, and
processed for serum, which was stored frozen at -70.degree. C.
Titer of virus in serum samples was determined by plaque assay on
Vero cells as described above. Neutralizing antibody titers were
determined for the day 28 serum samples as described above. A group
of monkeys inoculated with either 2A.DELTA.30 (n=4) or wt virus
814669 (n=8) were challenged on day 42 with a single dose of 5.0
log.sub.10PFU/ml wt virus 814669 and blood was collected for 10
days. Husbandry and care of rhesus monkeys was in accordance with
the National Institutes of Health guidelines for the humane use of
laboratory animals.
Construction and characterization of DEN-4 wild type and deletion
mutant viruses. The nucleotide and deduced amino acid sequences of
the previously described wt 814669 virus, the DEN-4 2A wt virus
derived from it (designated 2A wt), and the 2A.DELTA.30 vaccine
candidate derived from 2A wt virus were first determined. Sequence
analysis showed that the wt 814669 virus used in this study had
apparently accumulated 2 missense mutations (nucleotides 5826 and
7630) and 3 silent mutations during its passage and amplification
since these mutations were not described in previously published
reports of the viral sequence (GenBank accession number M14931) and
were not present in the 2A cDNA derived from the virus. Sequence
comparison between viruses 2A wt and vaccine lot 2A.DELTA.30
revealed that 2A.DELTA.30 accumulated 2 missense mutations
(nucleotides 7153 and 8308) and also confirmed the presence of the
.DELTA.30 mutation (nucleotides 10478-10507) as well as an
additional deletion of nucleotide 10475, which occurred during the
original construction of the .DELTA.30 mutation (Men, R. et al.
1996 J Virol 70:3930-7). This sequence analysis revealed
significant sequence divergence between the biologically-derived wt
814669 virus and its recombinant 2A wt derivative and between the
2A wt and 2A.DELTA.30 virus. Since the 2A wt and 2A.DELTA.30
viruses differed at nucleotides other than the deletion mutation,
the attenuation phenotype previously reported for 2A.DELTA.30 (Men,
R. et al. 1996 J Virol 70:3930-7) could not be formally ascribed
solely to the .DELTA.30 mutation and may have been specified by the
mutations at nucleotides 7153, 8308, 10475, or the .DELTA.30
deletion.
To determine whether the .DELTA.30 mutation was responsible for the
observed attenuation of 2A.DELTA.30, a second pair of viruses, one
with and one without the .DELTA.30 mutation, were produced for
evaluation in monkeys. A new DEN-4 cDNA vector construct,
designated p4, was derived from the 2A-XhoI cDNA clone and
translationally-silent mutations were introduced to add or ablate
several restriction enzyme sites. These sites were added to
facilitate the future genetic manipulation of this DEN-4 wt cDNA by
the introduction of other attenuating mutations if needed. The
sequence of the genomic region of the p4 cDNA plasmid was identical
to that of the 2A wt virus except for the engineered restriction
site changes and a point mutation at nucleotide 2440 which was
introduced during the original mutagenesis of the 2A cDNA plasmid
to create the XhoI site (Bray, M. & Lai, C. J. 1991 PNAS USA
88:10342-6). The .DELTA.30 mutation and the neighboring deletion at
nucleotide 10475 were co-introduced into the p4 plasmid by
replacing a short restriction fragment with one derived from the
cDNA clone of 2A.DELTA.30. RNA transcripts derived from the p4 cDNA
clone and from its .DELTA.30 derivative each yielded virus
(designated rDEN4 wt and rDEN4.DELTA.30, respectively) following
transfection of Vero cells. Sequence analysis of the rDEN4 virus
revealed that during its passage and amplification in Vero cells it
accumulated 2 missense mutations (nucleotides 4353 and 6195), a
silent mutation (nucleotide 10157), and a point mutation in the 3'
untranslated region (nucleotide 10452). In addition to containing
the .DELTA.30 and the accompanying deletion at nucleotide 10475,
rDEN4.DELTA.30 had also accumulated a missense mutation (nucleotide
7163) and a silent mutation (nucleotide 7295).
Parental wt 814669 virus and recombinant viruses 2A wt,
2A.DELTA.30, rDEN4, and rDEN4.DELTA.30 each replicate in Vero cells
to a titer exceeding 7.0 log.sub.10PFU/ml, and their replication is
not temperature sensitive at 39.degree. C.
Virus replication, immunogenicity, and efficacy in monkeys. Groups
of rhesus monkeys were inoculated with wt DEN-4 814669, 2A wt,
rDEN4, 2A.DELTA.30 and rDEN4.DELTA.30 to assess the level of
restriction of replication specified by the .DELTA.30 mutation.
Serum samples were collected daily and titer of virus present in
the serum was determined by plaque enumeration on Vero cell
monolayer cultures. Monkeys inoculated with wt 814669 virus or its
recombinant counterparts, 2A wt or rDEN4, were viremic for 3 to 4
days with a mean peak virus titer of nearly 2 log.sub.10PFU/ml.
Monkeys inoculated with virus 2A.DELTA.30 or rDEN4.DELTA.30 had a
lower frequency of viremia (83% and 50%, respectively), were
viremic for only about 1 day, and the mean peak titer was 10-fold
lower. Monkeys inoculated with DEN-4 814669, 2A wt, or rDEN4
viruses developed high levels of neutralizing antibody, with mean
titers between 442 and 532, consistent with their presumed wild
type phenotype. Monkeys inoculated with 2A.DELTA.30 or
rDEN4.DELTA.30 developed a lower level of neutralizing antibody,
with mean titers of 198 and 223, respectively. The decrease in
neutralizing antibody titer in response to 2A.DELTA.30 and
rDEN4.DELTA.30 is consistent with the attenuation phenotype of
these viruses. Monkeys inoculated with either 2A.DELTA.30 (n=4) or
wt 814669 virus (n=8) were challenged after 42 days with wt virus
814669. Dengue virus was not detected in any serum sample collected
for up to 10 days following virus challenge, indicating that these
monkeys were completely protected following immunization with
either wt virus or vaccine candidate 2A.DELTA.30.
Since DEN-4 814669, 2A wt, and rDEN4 each manifest the same level
of replication and immunogenicity in rhesus monkeys, it is
reasonable to conclude that the identified sequence differences
between these presumptive wild type viruses that arose during
passage in tissue culture or during plasmid construction do not
significantly affect their level of replication in vivo. Similarly,
the comparable level of attenuation of 2A.DELTA.30 and
rDEN4.DELTA.30 indicates that the mutations shared by these
viruses, namely, the .DELTA.30 mutation and its accompanying 10475
deletion mutation, are probably responsible for the attenuation of
these viruses rather than their incidental sequence
differences.
Clinical Response to immunization with 2A.DELTA.30. The 2A.DELTA.30
vaccine candidate was administered subcutaneously at a dose of
10.sup.5 PFU to 20 seronegative volunteers. Each of the vaccinees
was infected and the virus was well tolerated by all vaccinees.
Viremia was detected in 70% of the vaccinees, was present only at
low titer, and did not extend beyond day 11.
None of the 20 vaccinees reported soreness or swelling at the
injection site. Mild erythema (1-3 mm) around the injection site
was noted on examination of 8 volunteers 30 minutes
post-vaccination which resolved by the next day in 7 of those
volunteers and by the third day in the remaining volunteer. Mild
tenderness to pressure at the vaccination site was noted in 2
volunteers and lasted a maximum of 48 hours. During physical
examination, ten volunteers (50%) were noted to a have a very mild
dengue-like erythematous macular rash (truncal distribution) which
occurred with greatest frequency on day 10. None of the volunteers
noted the rash themselves, and it was asymptomatic in each
instance. Rash was seen only in vaccinees with detectable viremia.
Volunteers did not develop systemic illness. Seven volunteers noted
an occasional headache that was described as mild, lasting less
than 2 hours, and was not present in any volunteer on two
consecutive days. One volunteer reported fever of 38.6.degree. C.
and 38.2.degree. C. without accompanying headache, chills, eye
pain, photophobia, anorexia, myalgia, or arthralgia as an
outpatient the evening of day 3 and day 5, respectively. However,
this volunteer was afebrile when evaluated by the study staff on
the morning of days 3, 4, 5 and 6. All other temperature
measurements recorded by the volunteer or study staff were normal.
Although tourniquet tests were not performed, two volunteers were
noted to have petechiae at the site of the blood pressure cuff
after a blood pressure measurement was performed (one on day 6, the
other on days 7 and 10). Both of these volunteers had normal
platelet counts at that time and throughout the study.
Significant hematological abnormalities were not seen in any
vaccinee. Three vaccinees with presumed benign ethnic neutropenia
manifested an absolute neutrophil count (ANC) below 1500/mm.sup.3.
These three volunteers had baseline ANCs which were significantly
lower than the remaining 17 volunteers and which did not decrease
disproportionately to the other volunteers. Two of the three
volunteers who became neutropenic never had detectable viremia. A
mild increase in ALT levels was noted in 4 volunteers, and a more
significant increase in ALT level (up to 238 IU/L) was noted in one
volunteer. These ALT elevations were transient, were not associated
with hepatomegaly, and were completely asymptomatic in each of the
5 volunteers. Elevated ALT values returned to normal by day 26
post-vaccination. The volunteer with the high ALT value was also
noted to have an accompanying mild elevation in AST on day 14
(10.sup.4 IU/L) which also returned to baseline by day 26
post-vaccination. This volunteer did not have an associated
increase in LDH, bilirubin, or alkaline phosphatase levels.
Serologic response of humans to immunization with 2A.DELTA.30. Each
of the twenty vaccinees developed a significant rise in serum
neutralizing antibody titer against DEN-4 by day 28. The level of
serum neutralizing antibody was similar in viremic (1:662) and
non-viremic vaccinees (1:426). The DEN-4 neutralizing antibody
titers of both groups had not changed significantly by day 42.
Genetic stability of the .DELTA.30 mutation. RT-PCR and sequence
analysis of viral RNA isolated from serum samples (n=6) collected
from volunteers 6 to 10 days post-vaccination confirmed the
presence of the .DELTA.30 mutation and neighboring deletion at
nucleotide 10475.
Example 9
Pharmaceutical Compositions
Live attenuated dengue virus vaccines, using replicated virus of
the invention, are used for preventing or treating dengue virus
infection. Additionally, inactivated dengue virus vaccines are
provided by inactivating virus of the invention using known
methods, such as, but not limited to, formalin or
.beta.-propiolactone treatment. Live attenuated or inactivated
viruses containing the mutations described above form the basis of
an improved vaccine for the prevention or treatment of dengue
infection in humans.
Pharmaceutical compositions of the present invention comprise live
attenuated or inactivated dengue viruses, optionally further
comprising sterile aqueous or non-aqueous solutions, suspensions,
and emulsions. The composition can further comprise auxiliary
agents or excipients, as known in the art. See, e.g., Berkow et al.
eds. 1987 The Merck Manual, 15th edition, Merck and Co., Rahway,
N.J.; Goodman et al. eds. 1990 Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press,
Inc., Elmsford, N.Y.; Avery's Drug Treatment: Principles and
Practice of Clinical Pharmacology and Therapeutics, 3rd edition,
ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987; Osol,
A. ed. 1980 Remington's Pharmaceutical Sciences Mack Publishing Co,
Easton, Pa. pp. 1324-1341; Katzung, ed. 1992 Basic and Clinical
Pharmacology Fifth Edition, Appleton and Lange, Norwalk, Conn.
A virus vaccine composition of the present invention can comprise
from about 10.sup.2-10.sup.9 plaque forming units (PFU)/ml, or any
range or value therein, where the virus is attenuated. A vaccine
composition comprising an inactivated virus can comprise an amount
of virus corresponding to about 0.1 to 50 .mu.g of E protein/ml, or
any range or value therein.
The agents may be administered using techniques well known to those
in the art. Preferably, agents are formulated and administered
systemically. Suitable routes may include oral, rectal,
transmucosal, or intestinal administration; parenteral delivery,
including intramuscular, subcutaneous, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intraperitoneal, intradermal, intranasal, or intraocular
injections, just to name a few. For injection, the agents of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as saline, phosphate
buffered saline, Tris buffered saline, Hank's buffered saline,
growth media such as Eagle's Minimum Essential Medium (MEM), and
the like.
When a vaccine composition of the present invention is used for
administration to an individual, it can further comprise salts,
buffers, adjuvants, or other substances which are desirable for
improving the efficacy of the composition. Adjuvants useful with
the invention include, but are not limited to: (1) aluminum salts
(alum), such as aluminum hydroxide, aluminum phosphate, aluminum
sulfate, etc.; (2) oil-in-water emulsion formulations (with or
without other specific immunostimulating agents such as muramyl
peptides or bacterial cell wall components), such as for example
(a) MF59 (International Publication No. WO 90/14837), containing 5%
Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing
various amounts of MTP-PE, although not required) formulated into
submicron particles using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing
10% Squalene, 0.4% Tween 80, 5% pluronic -blocked polymer L121, and
thr-MDP either microfluidized into a submicron emulsion or vortexed
to generate a larger particle size emulsion, and (c) Ribi.TM.
adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)
containing 2% Squalene, 0.2% Tween 80, and one or more bacterial
cell wall components from the group consisting of
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (3) saponin
adjuvants, such as Stimulon.TM. (Cambridge Bioscience, Worcester,
Mass.) may be used or particle generated therefrom such as ISCOMs
(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA)
and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as
interleukins (IL-1, IL-2, etc.), macrophage colony stimulating
factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) mucosal
adjuvants such as those derived from cholera toxin (CT), pertussis
toxin (PT), E. coli heat labile toxin (LT), and mutants thereof
(see, e.g., International Publication Nos. WO 95/17211, WO
93/13202, and WO 97/02348); and (7) other substances that act as
immunostimulating agents to enhance the effectiveness of the
composition.
The pharmacologically active compounds of this invention can be
processed in accordance with conventional methods of galenic
pharmacy to produce medicinal agents for administration to
patients, e.g., mammals including humans.
The compounds of this invention can be employed in admixture with
conventional excipients, i.e., pharmaceutically acceptable organic
or inorganic carrier substances suitable for parenteral, enteral
(e.g., oral) or topical application, which do not deleteriously
react with the active compounds. Suitable pharmaceutically
acceptable carriers include but are not limited to water, salt
solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,
polyethylene glycols, gelatin, carbohydrates such as lactose,
amylose or starch, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume oil, fatty acid monoglycerides and diglycerides,
pentaerythritol fatty acid esters, hydroxy methylcellulose,
polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
coloring, flavoring and/or aromatic substances and the like which
do not deleteriously react with the active compounds. They can also
be combined where desired with other active agents, e.g.,
vitamins.
For parenteral application, particularly suitable are injectable,
sterile solutions, preferably oily or aqueous solutions, as well as
suspensions, emulsions, or implants, including suppositories.
Ampoules are convenient unit dosages.
For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules. A syrup,
elixir, or the like can be used wherein a sweetened vehicle is
employed.
For topical application, there are employed as non-sprayable forms,
viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, aerosols, etc., which are,
if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, stabilizers, wetting agents, buffers or salts for
influencing osmotic pressure, etc. For topical application, also
suitable are sprayable aerosol preparations wherein the active
ingredient, preferably in combination with a solid or liquid inert
carrier material, is packaged in a squeeze bottle or in admixture
with a pressurized volatile, normally gaseous propellant, e.g., a
freon.
The vaccine can also contain variable but small quantities of
endotoxin, free formaldehyde, and preservative, which have been
found safe and not contributing to the reactogenicity of the
vaccines for humans.
Example 10
Pharmaceutical Purposes
The administration of the vaccine composition may be for either a
"prophylactic" or "therapeutic" purpose. When provided
prophylactically, the compositions are provided before any symptom
of dengue viral infection becomes manifest. The prophylactic
administration of the composition serves to prevent or attenuate
any subsequent infection. When provided therapeutically, the live
attenuated or inactivated viral vaccine is provided upon the
detection of a symptom of actual infection. The therapeutic
administration of the compound(s) serves to attenuate any actual
infection. See, e.g., Berkow et al. eds. 1987 The Merck Manual,
15th edition, Merck and Co., Rahway, N.J.; Goodman et al. eds. 1990
Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th
edition, Pergamon Press, Inc., Elmsford, N.Y.; Avery's Drug
Treatment: Principles and Practice of Clinical Pharmacology and
Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,
Baltimore, Md. 1987; Katzung, ed. 1992 Basic and Clinical
Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn.
A live attenuated or inactivated vaccine composition of the present
invention may thus be provided either before the onset of infection
(so as to prevent or attenuate an anticipated infection) or after
the initiation of an actual infection.
The vaccines of the invention can be formulated according to known
methods to prepare pharmaceutically useful compositions, whereby
live attenuated or inactivated viruses are combined in a mixture
with a pharmaceutically acceptable vehicle. A composition is said
to be a "pharmacologically acceptable vehicle" if its
administration can be tolerated by a recipient patient. Suitable
vehicles are well known to those in the art, e.g., in Osol, A. ed.
1980 Remington's Pharmaceutical Sciences Mack Publishing Co,
Easton, Pa. pp. 1324-1341.
For purposes of administration, a vaccine composition of the
present invention is administered to a human recipient in a
therapeutically effective amount. Such an agent is said to be
administered in a "therapeutically effective amount" if the amount
administered is physiologically significant. A vaccine composition
of the present invention is physiologically significant if its
presence results in a detectable change in the physiology of a
recipient patient that generates a host immune response against at
least one dengue serotype, stimulates the production of
neutralizing antibodies, or leads to protection against
challenge.
The "protection" provided need not be absolute, i.e., the dengue
infection need not be totally prevented or eradicated, if there is
a statistically significant improvement compared with a control
population or set of patients. Protection may be limited to
mitigating the severity or rapidity of onset of symptoms of the
dengue virus infection.
Example 11
Pharmaceutical Administration
A vaccine of the present invention may confer resistance to one or
more dengue serotypes by immunization. In immunization, an live
attenuated or inactivated vaccine composition is administered
prophylactically, according to a method of the present invention.
In another embodiment a live attenuated or inactivated vaccine
composition is administered therapeutically, according to a
different method of the present invention.
The present invention thus includes methods for preventing or
attenuating infection by at least one dengue serotype. As used
herein, a vaccine is said to prevent or attenuate a disease if its
administration results either in the total or partial attenuation
(i.e., suppression) of a symptom or condition of the disease, or in
the total or partial immunity of the individual to the disease.
At least one live attenuated or inactivated dengue virus, or
composition thereof, of the present invention may be administered
by any means that achieve the intended purpose, using a
pharmaceutical composition as previously described.
For example, administration of such a composition may be by various
parenteral routes such as subcutaneous, intravenous, intradermal,
intramuscular, intraperitoneal, intranasal, oral or transdermal
routes. Parenteral administration can be by bolus injection or by
gradual perfusion over time. A preferred mode of using a
pharmaceutical composition of the present invention is by
intramuscular, intradermal or subcutaneous application. See, e.g.,
Berkow et al. eds. 1987 The Merck Manual 15th edition, Merck and
Co., Rahway, N.J.; Goodman et al. eds. 1990 Goodman and Gilman's
The Pharmacological Basis of Therapeutics, 8th edition, Pergamon
Press, Inc., Elmsford, N.Y; Avery's Drug Treatment: Principles and
Practice of Clinical Pharmacology and Therapeutics, 3rd edition,
ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987; Osol,
A. ed. 1980 Remington's Pharmaceutical Sciences, Mack Publishing
Co, Easton, Pa. pp. 1324-1341; Katzung, ed. 192 Basic and Clinical
Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn.
A typical regimen for preventing, suppressing, or treating a dengue
virus related pathology, comprises administration of an effective
amount of a vaccine composition as described herein, administered
as a single treatment, or repeated as enhancing or booster dosages,
over a period up to and including between one week and about 24
months, or any range or value therein.
It will be appreciated that the actual preferred amounts of active
compound in a specific case will vary according to the specific
compound being utilized, the compositions formulated, the mode of
application, and the particular situs and organism being treated.
Dosages for a given host can be determined using conventional
considerations, e.g., by customary comparison of the differential
activities of the subject compounds and of a known agent, e.g., by
means of an appropriate, conventional pharmacological protocol.
The dosage of a live attenuated virus vaccine for a mammalian
(e.g., human) subject can be from about 10.sup.3-10.sup.7 plaque
forming units (PFU)/kg, or any range or value therein. The dose of
inactivated vaccine can range from about 0.1 to 50 .mu.g of E
protein. However, the dosage should be a safe and effective amount
as determined by conventional methods, using existing vaccines as a
starting point.
The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
TABLE-US-00001 TABLE 1 Susceptibility of mice to intracerebral DEN4
infection is age-dependent.sup.a Mean virus titer (log.sub.10PFU/g
brain) .+-. SE following inoculation at indicated age (days) Virus
7 14 21 2A-13 >6.0 4.0 .+-. 0.2 3.1 .+-. 0.2 rDEN4 >6.0 3.3
.+-. 0.4 3.3 .+-. 0.2 rDEN4.DELTA.30 >6.0 3.6 .+-. 0.2 2.8 .+-.
0.3 .sup.aGroups of 4 or 5 Swiss Webster mice were inoculated
intracerebrally with 10.sup.5 PFU virus in a 30 .mu.l inoculum.
After 5 days, brains were removed, homogenized and titered in Vero
cells. SE = Standard error.
TABLE-US-00002 TABLE 2 Temperature-sensitive (ts) and mouse brain
attenuation (att) phenotypes of 5-FU mutant DEN4 viruses. Virus
replication in suckling mice.sup.b Mean virus titer
(log.sub.10PFU/ml) at indicated temp. (.degree. C.) Mean titer .+-.
SE Mean log.sub.10 Vero cells HuH-7 cells (log.sub.10PFU/g
reduction Phenotype Virus 35 37 38 39 .DELTA..sup.a 35 37 38 39
.DELTA. n brain) fro- m wt.sup.d wt (not ts) 2A-13 7.8 7.7 7.6 7.3
0.5 7.8 7.7 7.4 6.4 1.4 66 .sub. 6.6 .+-. 0.1.sup.c -- rDEN4 6.5
6.4 6.4 6.0 0.5 7.1 6.7 6.0 5.5 1.6 66 .sub. 6.1 .+-. 0.1.sup.c --
rDEN4.DELTA.30 6.3 6.1 6.1 5.7 0.6 6.9 6.3 5.9 4.7 2.2 64 .sub. 5.6
.+-. 0.1.sup.c 0.5 ts in Vero 695 6.2 6.0 5.2 2.6.sup.e 3.6 6.5 5.5
3.8 <1.6 >4.9 6 3.0 .+-. 0.2 3.2 and 816 6.8 6.4 5.8 3.9 2.9
7.5 6.2 5.5 3.1 4.4 6 3.3 .+-. 0.4 2.9 HuH-7 cells 773 7.4 6.6 6.0
3.1 4.3 7.7 6.1 5.2 3.1 4.6 12 3.7 .+-. 0.1 2.6 489 7.3 6.6 6.1 3.3
4.0 7.3 6.7 5.4 3.0 4.3 6 4.5 .+-. 0.5 2.3 173 7.0 6.1 3.2 2.9 4.1
7.0 3.2 3.0 2.1 4.9 18 4.7 .+-. 0.4 2.2 509 6.2 5.8 5.5 3.4 2.8 6.5
6.1 4.5 <1.6 >4.9 6 4.9 .+-. 0.3 1.9 938 7.1 6.5 5.6 3.1 4.0
7.2 6.4 5.6 3.1 4.1 6 5.1 .+-. 0.2 1.7 1033 6.7 6.0 5.9 4.1 2.6 6.9
5.6 4.7 <1.6 >5.3 12 4.7 .+-. 0.2 1.7 239 7.6 6.8 5.6 3.3 4.3
7.6 6.7 4.7 2.5 5.1 12 4.7 .+-. 0.3 1.5 793 6.5 5.8 5.3 4.0 2.5 7.2
6.8 5.6 <1.6 >5.6 6 5.4 .+-. 0.3 1.4 759 7.2 6.9 6.4 4.7 2.5
7.5 6.8 6.3 3.1 4.4 12 5.1 .+-. 0.1 1.4 718 6.1 5.9 5.3 3.5 2.6 7.0
6.5 5.7 1.7 5.3 12 5.0 .+-. 0.3 1.4 473 6.7 6.3 5.4 2.0 4.7 7.2 6.7
3.7 1.9 5.3 12 5.1 .+-. 0.3 1.2 ts in only 686 7.0 6.7 6.7 6.4 0.6
7.3 6.8 6.4 2.2 5.1 12 2.7 .+-. 0.2 3.8 HuH-7 cells 967 6.8 6.4 6.4
5.1 1.7 6.8 6.4 5.4 <1.6 >5.2 6 3.6 .+-. 0.2 2.9 992 7.3 7.1
6.8 5.9 1.4 7.4 6.9 5.0 <1.6 >5.8 6 3.8 .+-. 0.1 2.7 571 6.9
7.0 6.4 4.6 2.3 7.0 6.3 5.2 <1.6 >5.4 6 4.4 .+-. 0.4 2.4 605
7.6 7.5 7.1 6.9 0.7 7.8 7.2 6.8 <1.6 >6.2 12 4.5 .+-. 0.4 2.1
631 7.1 6.9 6.8 5.0 2.1 7.3 7.1 6.5 <1.6 >5.7 12 4.8 .+-. 0.3
1.9 1175 7.4 7.1 6.9 5.3 2.1 7.6 6.5 4.7 3.3 4.3 12 4.7 .+-. 0.2
1.7 .sup.aReduction in titer (lo.sub.g10PFU/ml) at 39.degree. C.
compared to titer at permissive temperature (35.degree. C.).
.sup.bGroups of 6 suckling mice were inoculated i.c. with 10.sup.4
PFU virus in a 30 .mu.l inoculum. Brains were removed 5 days later,
homogenized, and titered in Vero cells. .sup.cAverage of 11
experiments with a total of 64 to 66 mice per group.
.sup.dDetermined by comparing mean viral titers of mice inoculated
with mutant virus and the 2A-13 wt control in the same experiment
(n = 6 or 12). .sup.eUnderlined values indicate a 2.5 or 3.5
log.sub.10PFU/ml reduction in titer in Vero cells or HuH-7 cells,
respectively, at indicated temp when compared to titer at
permissive temp (35.degree. C.).
TABLE-US-00003 TABLE 3 Nucleotide and amino acid differences of the
5-FU mutant viruses which are ts in both Vero and HuH-7 cells.
Mutations in UTR or Mutations in coding region coding region that
result in that do not result in an an amino acid substitution amino
acid substitution Nucleotide Gene/ Nucleotide Amino Acid Nucleotide
Nucleotide Virus position region change change.sup.b position Gene
change 173.sup.a 7163 NS4B A > C L2354F 10217 NS5 A > U 7849
NS5 A > U N2583I 8872 NS5 A > G K2924R 239.sup.a 4995 NS3 U
> C S1632P 7511 NS4B G > A 10070 NS5 U > C 473.sup.a 4480
NS2B U > C V1460A 7589 NS5 G > A 4995 NS3 U > C S1632P
10070 NS5 U > C 489.sup.a 4995 NS3 U > C S1632P 2232 E U >
C 3737 NS2A C > U 509.sup.a 4266 NS2B A > G S1389G none 8092
NS5 A > G E2664G 695.sub. 40 5' UTR U > C n/a 1391 E A > G
1455 E G > U V452F 6106 NS3 A > G E2002G 7546 NS4B C > U
A2482V 718.sub. 2280 E U > C F727L none 4059 NS2A A > G
I1320V 4995 NS3 U > C S1632P 7630 NS5 A > G K2510R 8281 NS5 U
> C L2727S 759.sup.a 4995 NS3 U > C S1632P none 8020 NS5 A
> U N2640I 773.sup.a 4995 NS3 U > C S1632P none 793.sub. 1776
E G > A A559T 5771 NS3 U > C 2596 NS1 G > A R832K 7793 NS5
U > A 2677 NS1 A > G D859G 4387 NS2B C > U S1429F
816.sup.a 4995 NS3 U > C S1632P 6632 NS4A G > A 7174 NS4B C
> U A2358V 6695 NS4A G > A 938.sup.a 3442 NS1 A >G E1114G
747 prM U > C 4995 NS3 U > C S1632P 4196 NS2b U > C 10275
3' UTR A > U n/a 6155 NS3 G >A 1033.sup.a 4907 NS3 A > U
L1602F 548 prM C > U 8730 NS5 A > C N2877H 9977 NS5 G > A
M3292I .sup.aViruses that contain mutation(s) resulting in an a.a.
substitution in only a NS gene(s) and/or nucleotide substitutions
in the UTRs are indicated; i.e. no a.a. substitutions are present
in the structural proteins (C-prM-E). .sup.bAmino acid position in
DEN4 polyprotein beginning with the methionine residue of the C
protein (nt 102-104) as residue #1. Wild-type amino acid on left of
amino acid position; mutant amino acid on right.
TABLE-US-00004 TABLE 4 Nucleotide and amino acid differences of the
5-FU mutant viruses which are ts in only HuH-7 cells. Mutations in
UTR or coding Mutations in coding region region that result in an
that do not result in an amino acid substitution amino acid
substitution Nucleotide Gene/ Nucleotide Amino acid Nucleotide
Nucleotide Virus position region change changeb position Gene
change 571 586 prM U > C V162A 6413 NS4A U > C 7163 NS4B A
> U L2354F 7947 NS5 G > A G2616R 605 1455 E G > U V452F
none 7546 NS4B C > U A2482V 631 595 prM A > G K165R 1175 E G
> A 6259 NS3 U > C V2053A 5174 NS3 A > G 7546 NS4B C >
U A2482V .sub. 686.sup.a 3575 NS2A G > A M1158I 4604 NS3 A >
G 4062 NS2A A > G T1321A 7937 NS5 A > G 7163 NS4B A > U
L2354F 967 2094 E G > C A665P 4616 NS3 C > U 2416 E U > C
V772A 7162 NS4B U > C L2354S 7881 NS5 G > A G2594S .sub.
992.sup.a 5695 NS3 A > G D1865G 3542 NS2A A > G 7162 NS4B U
> C L2354S 1175.sup.a 7153 NS4B U > C V2351A 6167 NS3 U >
C 10186 NS5 U > C I3362T 10184 NS5 G >A 10275 3' UTR A > U
n/a .sup.aViruses that contain mutation(s) resulting in an a.a.
substitution in only a NS gene(s) and/or nucleotide substitutions
in the UTRs are indicated; i.e. no a.a. substitutions are present
in the structural proteins. .sup.bAmino acid position in DEN4
polyprotein beginning with the methionine residue of the C protein
(nt 102-104) as residue #1. Wild-type amino acid on left of amino
acid position; mutant amino acid on right.
TABLE-US-00005 TABLE 5 Mutations which are represented in multiple
5-FU mutant DEN4 viruses. Nucleotide Gene/ Nucleotide Amino acid
Number of viruses position region change change with "sister"
mutations 1455 E G > U val > phe 2 4995 NS3 U > C ser >
pro 8 7162 NS4B U > C leu > ser 2 7163 NS4B A > U or C leu
> phe 3 7546 NS4B C > U ala > val 3 10275 3' UTR A > U
n/a.sup.a 2 .sup.anot applicable
TABLE-US-00006 TABLE 6 Addition of ts mutation 4995 to
rDEN4.DELTA.30 confers a ts phenotype and further attenuates its
replication in suckling mouse brain. Replication in suckling
mice.sup.b Mean virus titer (log.sub.10PFU/ml) Mean virus at
indicated temp (.degree. C.) titer .+-. SE Mean log.sub.10 Vero
cells HuH-7 cells (log.sub.10PFU/g reduction Virus 35 37 38 39
.DELTA..sup.a 35 37 38 39 .DELTA. brain) from wt.sup.c 2A-13 7.1
7.1 6.9 6.8 0.3 7.4 7.3 6.7 6.4 1.0 6.5 .+-. 0.1 -- rDEN4 7.0 6.8
6.6 6.4 0.6 7.5 7.3 6.7 6.4 1.1 6.1 .+-. 0.2 -- rDEN4.DELTA.30 7.0
6.7 6.2 6.2 0.8 7.5 7.0 6.5 5.1 2.4 5.9 .+-. 0.1 0.2 rDEN4-4995 5.7
4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 3.2 .+-. 0.2
2.9 rDEN4.DELTA.30-4995 5.9 4.9 3.9 <1.6.sup.d >4.3 6.4 5.6
4.4 <1.6 - >4.8 3.0 .+-. 0.3 3.1 .sup.aReduction in titer
(log.sub.10PFU/ml) at 39.degree. C. compared to titer at permissive
temperature (35.degree. C.). .sup.bGroups of 6 suckling mice were
inoculated i.c. with 10.sup.4 PFU virus in a 30 .mu.l inoculum.
Brains were removed 5 days later, homogenized, and titered in Vero
cells. The limit of detection is 2.0 log.sub.10PFU/g brain.
.sup.cDetermined by comparing mean viral titers of mice inoculated
with sample virus and rDEN4 control. .sup.dUnderlined values
indicate a 2.5 or 3.5 log.sub.10PFU/ml reduction in titer in Vero
cells or HuH-7 cells, respectively, at indicated temperature when
compared to permissive temperature.
TABLE-US-00007 TABLE 7 Temperature-sensitive (ts) and mouse brain
attenuation (att) phenotypes of 5-FU DEN4 mutant viruses which
exhibit a small plaque (sp) phenotype. Replication in suckling
mice.sup.b Phenotype Mean virus titer (log.sub.10PFU/ml) Mean virus
Mean sp ts at indicated temp (.degree. C.) titer .+-. SE log.sub.10
HuH- HuH- Vero cells HuH-7 cells (log.sub.10PFU/ reduction Vero 7
Vero 7 Virus 35 37 38 39 .DELTA..sup.a 35 37 38 39 .DELTA. n g
brain) from wt.sup.d - - - - 2A-13 7.9 7.5 7.7 7.2 0.7 7.9 7.7 7.3
6.9 1.0 66 .sub. 6.6 .+-. 0.1.sup.c -- - - - - rDEN4 7.9 7.6 7.7
7.3 0.6 8.1 7.6 7.5 6.7 1.4 66 .sub. 6.1 .+-. 0.1.sup.c -- - - - -
rDEN4.DELTA.30 7.3 6.6 6.6 6.1 1.2 7.3 7.2 6.9 5.9 1.4 64 .sub. 5.6
.+-. 0.1.sup.c 0.5 + + + + 574 6.6.sup.x 5.5 3.8 <1.6.sup.e
.gtoreq.5.0 6.6.sup.x 4.9 5.0 - <1.6 .gtoreq.5.0 6 2.1 .+-. 0.1
5.1 + + + + 1,269 5.3.sup.x 4.8 3.9 <1.6 .gtoreq.3.7 4.0.sup.x
2.4 2.0 <- 1.6 .gtoreq.2.4 6 2.7 .+-. 0.2 4.1 + + + + 1,189
6.3.sup.x 5.2 4.5 3.8 2.5 5.5.sup.x 3.7 2.3 <1.6 .gtoreq.- 3.9
12 3.2 .+-. 0.4 3.7 + + - - 569 5.8.sup.x 5.6 5.6 3.7 2.1 6.2.sup.x
6.0 5.7 5.0 1.2 12 1.9 .+-. 0.1 4.6 + + - - 761 5.0.sup.x 4.7 4.2
2.7 2.3 5.6.sup.x 5.3 4.5 2.6 3.0 12 2.0 .+-. 0.1 4.2 - + + + 506
7.0 6.8 5.6 2.6 4.4 6.7.sup.x 4.3 <1.6 2.0 4.7 6 2.2 .+-. 0.1
4.7 - + + + 1,136 5.1 4.2 2.6 <1.6 .gtoreq.3.5 5.7.sup.x 3.0 3.0
<1.6 .g- toreq.4.1 6 2.9 .+-. 0.3 4.5 - + + + 1,029 6.9 5.8 5.8
2.9 4.0 7.0.sup.x 5.8 5.2 2.5 4.5 6 2.2 .+-. 0.1 4.2 - + + + 1,081
6.9 5.8 4.7 3.9 3.0 5.8.sup.x 4.1 3.3 1.9 3.9 12 2.6 .+-. 0.2 3.9 -
+ + + 529 6.9 6.5 5.9 4.0 2.9 7.1.sup.x 5.3 4.4 <1.6 .gtoreq.5.5
6 3.- 1 .+-. 0.7 3.8 - + + + 1,114 6.7 6.4 6.2 2.5 4.2 5.7.sup.x
3.0 2.9 1.9 3.8 6 2.7 .+-. 0.3 3.7 - + + + 922 7.3 7.2 6.8 3.8 3.5
7.4.sup.x 5.3 4.1 3.0 4.4 12 3.5 .+-. 0.1 2.9 - + + + 311 6.9 5.9
4.3 1.5 5.4 7.1.sup.x 5.4 3.6 <1.6 .gtoreq.5.5 12 6- .1 .+-. 0.3
0.9 - + + + 326 6.6 5.7 4.5 3.1 3.5 7.0.sup.x 5.5 4.1 2.0 5.0 6 6.0
.+-. 0.1 0.9 - + - + 1,104 7.1 6.8 6.8 6.1 1.0 7.2.sup.x 6.4 5.8
2.8 4.4 6 2.2 .+-. 0.1 4.7 - + - + 952 7.1 7.0 6.7 5.6 1.5
7.3.sup.x 6.3 5.6 3.0 4.3 6 2.4 .+-. 0.3 4.5 - + - + 738 6.5 6.0
5.9 5.7 0.8 6.9.sup.x 6.1 5.0 3.1 3.8 12 4.4 .+-. 0.4 2.3 - + - +
1,083 7.4 7.3 7.4 5.8 1.6 7.4.sup.x 6.6 4.5 <1.6 .gtoreq.5.8 12-
4.5 .+-. 0.4 2.0 - + - - 1,096 7.5 7.1 6.9 5.5 2.0 7.5.sup.x 6.6
5.6 4.8 2.7 6 2.9 .+-. 0.2 3.5 - + - - 1,021 7.0 6.9 6.6 6.3 0.7
6.9.sup.x 5.7 4.4 4.0 2.9 6 3.9 .+-. 0.6 2.6 - + - - 1,023 6.6 6.4
6.0 5.8 0.8 6.1.sup.x 5.6 4.7 3.3 2.8 12 4.2 .+-. 0.3 2.3 - + - -
1,012 7.5 7.1 7.0 5.7 1.8 7.4.sup.x 6.8 6.8 5.6 1.8 6 6.1 .+-. 0.1
0.8 .sup.aReduction in mean virus titer (log.sub.10PFU/ml) at
39.degree. C. compared to permissive temperature (35.degree. C.).
.sup.bGroups of 6 suckling mice were inoculated i.c. with 10.sup.4
PFU virus. Brains were removed 5 days later, homogenized, and
titered in Vero cells. .sup.cAverage of 11 experiments with a total
of 64 to 66 mice per group. .sup.dDetermined by comparing mean
viral titers of mice inoculated with mutant virus and concurrent
2A-13 wild type (wt) virus control (n = 6 or 12). .sup.eUnderlined
values indicate a 2.5 or 3.5 log.sub.10PFU/ml reduction in titer in
Vero cells or HuH-7 cells, respectively, at indicated temperature
when compared to permissive temperature (35.degree. C.). xSmall
plaque size at 35.degree. C.; small plaques have a diameter of
<1.0 mm compared to wild type plaque diameter of 1.5-2.0 mm in
Vero cells, or a diameter of <0.4 mm compared to wild type
plaque diameter of 0.75 to 1.0 mm in HuH-7 cells.
TABLE-US-00008 TABLE 8 Viruses with both ts and sp phenotypes are
more restricted in replication in mouse brain than those with only
a ts phenotype. Cell culture Number Mean log.sub.10 reduction in
virus titer phenotype of viruses from controls.sup.b,c ts.sup.a 20
2.1 .+-. 0.2 sp 6 3.0 .+-. 0.6 ts/sp 16 3.5 .+-. 0.3 .sup.a20 ts
mutant viruses without an sp phenotype were previously described
(Example 1). .sup.bDetermined by comparing mean viral titers of
groups of mice inoculated with mutant virus and concurrent 2A-13
parallel-passaged control virus. .sup.cSignificant difference
between ts group and ts/sp group, Tukey-Kramer test (P <
0.05)
TABLE-US-00009 TABLE 9 Nucleotide and amino acid differences of the
5-FU mutant DEN4 viruses which produce small plaques in both Vero
and HuH-7 cells. Mutations in UTR or in coding regions Mutations in
coding regions that do not that result in an amino acid
substitution result in an amino acid substitution Nucleotide Gene/
Nucleotide Amino acid Nucleotide Nucleotide Virus position region
change change.sup.b position Gene change 569 826 prM G > A R242K
1946 E C > U 832 prM C > U P244L 7546 NS4B C > U A2482V
10275 3 ' UTR A > U n/a 10279 3 ' UTR A > U n/a 574 1455 E G
> U V452F 1349 E C > U 1963 E U > C V621A 3880 NS2A A >
G K1260R 7546 NS4B C > U A2482V 7615 NS5 A > G N2505S 10413 3
' UTR A > G n/a 761 424 C U > C I108T none 2280 E U > C
F727L 7131 NS4B A > G T2344A 7486 NS4B A > G N2462S 1189a
3303 NS1 A > G R1068G 6719 NS4A U > C 4812 NS3 G > A
V1571I 5097 NS3 G > A D1666N 7182 NS4B G > A G2361S 1269 2112
E U > C F671L 542 prM C > U 3256 NS1 G > A G1052E 3993
NS2A U > C F1298L 7183 NS4B G > U G2361V .sup.aVirus contains
missense mutations in only the non-structural genes. .sup.bAmino
acid position in DEN4 polyprotein beginning with the methionine
residue of the C protein (nt 102-104). Wild type amino acid on left
of amino acid position; mutant amino acid on right.
TABLE-US-00010 TABLE 10 Nucleotide and amino acid differences of
the 5-FU mutant DEN4 viruses which produce small plaques in only
HuH-7 cells. Mutations in UTR or in Mutations in coding regions
coding regions that result that do not result in an amino in an
amino acid substitution acid substitution Nucleotide Gene/
Nucleotide Amino acid Nucleotide Nucleotide Virus position region
change change.sup.b position Gene change 311 1519 E A > G N473S
6761 NS4A C > U 2305 E G > A R735K 10070 NS5 U > C 4896
NS3 G > U A1599S 326 1587 E C > U P496S 1523 E G > A 7546
NS4B C > U A2482V 6080 NS3 U > C 10070 NS5 U > C 506 1455
E G > U V452F 3887 NS2A A > G 1902 E G > A V601M 5789 NS3
G > C 7546 NS4B C > U A2482V 10275 3 ' UTR A > U n/a 529
777 prM U > C S226P none 4641 NS3 A > G I1514V 7153 NS4B U
> C V2351A 8245 NS5 U > C I2715T 10279 3 ' UTR A > C n/a
.sup. 738.sup.a 3540 NS2A G > A E1147K none 7162 NS4B U > C
L2354S .sup. 922.sup.a 4306 NS2B A > G N1402S 7736 NS5 G > A
5872 NS3 C > U T1924I 7163 NS4B A > U L2354F 10279 3 ' UTR A
> C n/a 952 1449 E G > U V450L none 1455 E G > U V452F
7546 NS4B C > U A2482V 7957 NS5 U > C V2619A 9543 NS5 A >
G I3148V 1012 1542 E A > G K481E 953 E A > G 7162 NS4B U >
C L2354S 1205 E G > A 10542 3 ' UTR A > G n/a 4425 NS2B U
> C 1021 2314 E U > C I738T 665 prM C > A 3205 NS1 C >
U A1035V 5750 NS3 C > U 4029 NS2A U > C C1310R 9959 NS5 C
> U 7163 NS4B A > C L2354F 10275 3 ' UTR A > U n/a 10279 3
' UTR A > U n/a 1023 2283 E G > A G728R 1001 E C > U 7182
NS4B G > A G2361S 1958 E A > G 3873 NS2a U > C 8486 NS5 C
> U 1029 850 prM C > U A250V 3867 NS2a C > U 3087 NS1 A
> G T996A 4891 NS3 U > C I1597T .sup. 1081.sup.a 2650 NS1 A
> G N850S 6326 NS3 C > U 7163 NS4B A > U L2354F 9146 NS5 C
> U .sup. 1083.sup.a 3702 NS2A G > A A1201T 3353 NS1 A > G
7153 NS4B U > C V2351A 6155 NS3 G > A 10634 3 ' UTR U > C
n/a 1096 892 prM G > A R264Q 665 prM C > A 7163 NS4B A > C
L2354F 4427 NS2b G > A 8659 NS5 C > U P2853L 1104 1692 E G
> A V531M none 5779 NS3 C > U A1893V 7546 NS4B C > U
A2482V 1114 709 prM A > G K203R 1076 E U > C 3693 NS2A A >
G I1198V 1182 E C > U 4614 NS3 U > C F1505L 5690 NS3 C > U
7546 NS4B C > U A2482V 9942 NS5 A > G T3281A .sup. 1136.sup.a
3771 NS2A A > G R1224G 5621 NS3 A > G 4891 NS3 U > C
I1597T 10275 3 ' UTR A > U n/a .sup.aViruses that contain
missense mutations in only the non-structural genes and/or
mutations in the UTRs. .sup.bAmino acid position in DEN4
polyprotein beginning with the methionine residue of the C protein
(nt 102-104). Wild type amino acid on left of amino acid position;
mutant amino acid on right.
TABLE-US-00011 TABLE 11 Putative Vero cell adaptation mutations
derived from the full set of 5-FU mutant viruses. 5-FU mutant
viruses Nucleotide Gene/region Nucleotide Amino acid No. of viruses
position (a.a. #).sup.b change change with the mutation 1455 E
(452) G > U Val > Phe 5 2280 E (727) U > C Phe > Leu 2
4891 NS3 (1597) U > C Ile > Thr 2 4995 NS3 (1599) U > C
Ser > Pro 8 7153 NS4B (2351) U > C Val > Ala 3 7162 NS4B
(2354) U > C Leu > Ser 4 7163 NS4B (2354) A > U or C Leu
> Phe 7 7182 NS4B (2361) G > A Gly > Ser 2 7546 NS4B
(2482) C > U Ala > Val 10 7630 NS5 (2510) A > G Lys >
Arg 1 10275 3' UTR A > U n/a.sup.a 6 10279 3' UTR A > C n/a 4
.sup.anot applicable .sup.bAmino acid position in DEN4 polyprotein
beginning with the methionine residue of the C protein (nt 102-104)
as residue #1.
TABLE-US-00012 TABLE 12 Mutagenic oligonucleotides used to generate
recombinant DEN4 viruses containing single 5-FU mutations. SEQ
Recombinant Amino ID virus Nucleotide acid pUC RE NO. (rDEN4-)
change change Gene clone site.sup.a Oligonucleotide.sup.b 23 40 U
> C n/a 5 ' UTR pUC-NheI BsaWI CAGTTCCAAAcCGGAAGCTTG 24 2650 A
> G Asn > Ser NS1 pUC-NS1 BsiWI CCAACGAGCTAtcgTAcGTTCTCTGGG
25 3303 A > G Arg > Gly NS1 pUC-NS1 StyI
GATTGTGACCATgGcGGCCCATCTTTG 26 3442 A > G Glu > Gly NS1
pUC-NS1 BlpI GGAGATTAGGCCgcTGAGcGgtAAAGAAGAG 27 3540 G > A Glu
> Lys NS2A pUC-NS1 BsmI GTTTGTGGAAaAATGtcTGAGGAGAA 28 3575 G
> A Met > Ile NS2A pUC-NS1 SspI CTAGGAAACACATaATATTAGTTGTGG
29 3702 G > A Ala > Thr NS2A pUC-NS2A BglI
CAGATCCACCTAaCCATaATGGCAGTG 30 3771 A > G Arg > Gly NS2A
pUC-NS2A AvaI GGAAACTCACcTCggGAGAGACAGC 31 4059 A > G Ile >
Val NS2A pUC-NS2A BstEII TTGGGTAGAggTcACcGCACTCATCC 32 4062 A >
G Thr > Ala NS2A pUC-NS2A BsrBI GTAGAAATAgCcGCtCTCATCCTAG 33
4266 A > G Ser > Gly NS2B pUC-NS2A SnaBI
GGCGGCTTACGTaATGgGaGGTAGCTCAGC 34 4306 A > G Asn > Ser NS2B
pUC-NS2A A1wNI CTAGAGAAGGCaGCttctGTGCAGTGG 35 4480 U > C Val
> Ala NS2B pUC-NS2A MscI CCTTGGCcATTCCAGcaACAATGAC 36 4812 G
> A Val > Ile NS3 pUC-NS2A ApoI GACGTTCAaaTttTaGCCATAGAACC 37
4891 U > C Ile > Thr NS3 pUC-NS2A KasI
CTGGAGAAAcgGGcGCcGTAACATTAG 38 4896 G > U Ala > Ser NS3
pUC-NS2A BstEII GAAATTGGAtCgGTAACcTTAGATTTC 39 4907 A > U Leu
> Phe NS3 pUC-NS2A AclI GGAGCAGTAACgTTtGATTTCAAACCC 40 4995 U
> C Ser > Pro NS3 pUC-NS2A BsaJI GTTACCAAAcCtGGgGATTACGTC 41
5097 G > A Asp > Asn NS3 pUC-NS3 BspHI
GATTAACTATcATGaACTTACACCC 42 5695 A > G Asp > Gly NS3 pUC-NS3
BanI GGAAAACCTTTGgcACcGAGTATCC 43 5872 C > U Thr > Ile NS3
pUC-NS3 BsrFI TCCAGTGAtaCCgGCtAGCGCTGCTC 44 6106 A > G Glu >
Gly NS3 pUC-NS3 MscI GCCTCAGAGGtGgcCAAAGGAAG 45 6259 U > C Val
> Ala NS3 pUC-NS3 BglII ACATGGAGGcaGAgATcTGGACTAGA 46 7153 U
> C Val > Ala NS4B pUC-NS4A MscI AAAGCATGgCcAAGGATGCTGTC 47
7162 U > C Leu > Ser NS4B pUC-NS4A BlpI
GCATAATGGACgctAAGCATGACTAAGG 48 7163 A > C Leu > Phe NS4B
pUC-NS4A ApaLI TTATTGCATAgTGcACgAAAAGCATG 49 7174 C > U Ala >
Val NS4B pUC-NS4A BsaAI GGGCCTATTATTaCgTAATGGAC 50 7182 G > A
Gly > Ser NS4B pUC-NS4A n/a CTGCAATCCTGGtgaTATTATTGC 51 7546 C
> U Ala > Val NS4B pUC-NSSA AclI CTCATAAAGAAcGttCAAACCCT 52
7630 A > G Lys > Arg NS5 pUC-NSSA HgaI
CATTAGACAGAcgcGAGTTTGAAG 53 7849 A > U Asn > Ile NS5 pUC-NSSA
HgaI TGGCGACgCTCAAGAtaGTGACTGAAG 54 8020 A > U Asn > Ile NS5
pUC-NSSA ClaI GAGTCATCaTCgAtaCCAACAATAG 55 8092 A > G Glu >
Gly NS5 pUC-NSSA EcoRI CTTCAAAACCTGgcTTCTGCATCAAAG 56 8281 U > C
Leu > Ser NS5 pUC-NSSB XmnI CAAAGATGTTGagcAACAGGTTCACAAC 57 8730
A > C Asn > His NS5 pUC-NSSB AvaI GGAAAGAAGAAAcAcCCgAGACTGTGC
58 8872 A > G Lys > Arg NS5 pUC-NSSB PvuI
GGGAACTGGTcGAtcgAGAAAGGGC 59 9977 G > A Met > Ile NS5
pUC-NSSC SfcI CCAGTGGATtACtACaGAAGATATGCTC 60 10186 U > C Ile
> Thr NS5 pUC-NSSC AgeI CAGGAACCTGAcCGGtAAAGAGGAATACG 61 10275 A
> U n/a 3 ' UTR pUC-NSSC n/a CTGTAATTACCAACAtCAAACACCAAAG 62
10279 A > C n/a 3 ' UTR pUC-NSSC n/a CCAACAACAAcCACCAAAGGCTATTG
63 10634 U > C n/a 3 ' UTR pUC-3' UTR n/a
GGATTGGTGTTGTcGATCCAACAGG .sup.aPrimers were engineered which
introduced (underline) or ablated (hatched line)
translationally-silent restriction enzyme sites. .sup.bLowercase
letters indicate nt changes and bold letters indicate the site of
the 5-FU mutation, which in some oligonucleotides differs from the
original nucleotide substitution change in order to create a unique
restriction enzyme site. The change preserves the codon for the
amino acid substitution.
TABLE-US-00013 TABLE 13 sp, ts and mouse attenuation phenotypes of
rDEN4 mutant viruses encoding single mutations identified in six sp
5-FU mutant viruses. Replication in Replication in suckling
HuH-7-SCID mice.sup.d Mean virus titer mice.sup.b Mean peak Mean
log.sub.10- 5-FU Gene/ (log.sub.10PFU/ml) at Mean virus Mean virus
titer .+-. unit mu- region indicated temp (.degree. C.) titer .+-.
SE log.sub.10-unit SE (log.sub.10 reduction tant containing Vero
cells HuH-7 cells (log.sub.10PFU/ reduction from PFU/ml from value
virus Virus mutation 35 39 .DELTA..sup.a 35 39 .DELTA. n g brain)
value for wt.sup.c n serum) for wt.sup.c 2A-13 7.6 7.1 0.5 7.8 6.6
1.2 30 6.5 .+-. 0.1 -- 29 6.8 .+-. 0.2 -- rDEN4 7.6 6.8 0.8 8.0 6.7
1.3 54 5.8 .+-. 0.1 -- 32 6.3 .+-. 0.2 -- rDEN4.DELTA.30 7.6 6.9
0.7 7.7 5.6 2.1 30 5.6 .+-. 0.1 0.2 18 5.4 .+-. 0.2 0.9 738 parent
6.5 5.7 0.8 .sup.x6.9 3.1.sup.e 3.8 12 4.4 .+-. 0.4 2.3 9 5.4 .+-.
0.7 1.9 rDEN4-3540 NS2A 6.9 5.1 1.8 7.4 3.7 3.7 12 4.1 .+-. 0.3 1.7
5 6.1 .+-. 0.3 (+)0.1 rDEN4-7162 NS4B 7.2 6.8 0.4 7.4 6.6 0.8 8 5.6
.+-. 0.3 0.3 5 6.8 .+-. 0.6 0.3 922 parent 7.3 3.8 3.5 .sup.x7.4
3.0 4.4 12 3.5 .+-. 0.1 2.9 6 6.2 .+-. 0.2 0.4 rDEN4-4306 NS2B
.sup.x5.0 2.2 2.8 .sup.x5.6 <1.6 >4.0 12 1.7 .+-. 0.1 4.1 5
5.2 .+-. 0.6 1.1 rDEN4-5872 NS3 5.7 2.5 3.2 .sup.x6.5 <1.6
>4.9 12 4.5 .+-. 0.3 1.3 5 6.2 .+-. 0.5 0.1 rDEN4-7163 NS4B 7.8
7.2 0.6 8.0 7.4 0.6 6 6.2 .+-. 0.2 (+)0.1 6 5.8 .+-. 0.6 (+)0.2
rDEN4-10279 3 ' UTR 6.9 5.7 1.2 7.7 5.7 2.0 6 4.8 .+-. 0.2 0.7 4
6.7 .+-. 0.2 0.4 1081 parent 6.9 3.9 3.0 .sup.x5.8 1.9 3.9 12 2.6
.+-. 0.2 3.9 4 4.2 .+-. 0.5 2.4 rDEN4-2650 NS1 5.1 3.0 2.1
.sup.x5.5 2.8 2.7 12 3.0 .+-. 0.3 2.8 6 4.7 .+-. 0.5 2.2 rDEN4-7163
NS4B 7.8 7.2 0.6 8.0 7.4 0.6 6 6.2 .+-. 0.2 (+)0.1 6 5.8 .+-. 0.6
(+)0.2 1083 parent 7.4 5.8 1.6 .sup.x7.4 <1.6 .gtoreq.5.8 12 4.5
.+-. 0.4 2.0 9 4.4 .+-. 0.3 2.9 rDEN4-3702 NS2A 6.8 5.6 1.2 7.6 4.7
2.9 18 4.9 .+-. 0.3 0.9 7 6.3 .+-. 0.3 0.2 rDEN4-7153 NS4B 7.7 7.2
0.5 8.0 6.9 1.1 6 5.7 .+-. 0.1 0.2 4 5.9 .+-. 0.7 0.1 rDEN4-10634 3
' UTR 4.9 1.6 3.3 .sup.x5.7 <1.6 .gtoreq.4.1 12 2.4 .+-. 0.3 3.4
7 3.3 .+-. 0.4 3.6 1136 parent 5.1 <1.6 .gtoreq.3.5 .sup.x5.7
<1.6 .gtoreq.4.1 6 2.9 .+-. 0.3 4.5 7 4.5 .+-. 0.4 1.2
rDEN4-3771 NS2A 7.0 4.6 2.4 .sup.x7.6 3.7 3.9 12 2.6 .+-. 0.4 3.2 4
6.4 .+-. 0.2 (+)0.1 rDEN4-4891 NS3 7.1 <1.6 >5.5 .sup.x7.4
<1.6 >5.8 12 2.5 .+-. 0.3 3.5 6 6.0 .+-. 0.5 0.3 rDEN4-10275
3 ' UTR 6.9 5.8 1.1 7.1 5.2 1.9 6 5.0 .+-. 0.3 0.5 4 6.7 .+-. 0.3
0.4 1189 parent .sup.x6.3 3.8 2.5 .sup.x5.5 <1.6 .gtoreq.3.9 12
3.2 .+-. 0.4 3.7 13 2.3 .+-. 0.3 3.8 rDEN4-3303 NS1 6.1 4.8 1.3 6.6
3.9 2.7 8 5.7 .+-. 0.4 0.2 4 6.3 .+-. 0.3 0.8 rDEN4-4812 NS3 7.0
6.3 0.7 7.1 6.3 0.8 12 4.8 .+-. 0.2 1.0 5 6.1 .+-. 0.5 (+)0.5
rDEN4-5097 NS3 .sup.x5.0 <1.6 >3.4 .sup.x4.6 <1.6 >3.0
12 1.8 .+-. 0.1 4.0 8 1.9 .+-. 0.1 4.3 rDEN4-7182 NS4B 7.7 6.9 0.8
7.8 6.8 1.0 6 6.2 .+-. 0.1 (+)0.1 6 6.3 .+-. 0.3 (+)0.7
.sup.aReduction in mean virus titer (log.sub.10PFU/ml) at
39.degree. C. compared to permissive temperature (35.degree. C.).
.sup.bGroups of 6 suckling mice were inoculated i.c. with 10.sup.4
PFU of virus. Brains were removed 5 days later, homogenized, and
titered in Vero cells. .sup.cComparison of mean virus titers of
mice inoculated with mutant virus and concurrent DEN4 control. Bold
denotes .gtoreq.50- or .gtoreq.100- fold decrease in replication in
suckling or SCID-HuH-7 mice, respectively. .sup.dGroups of
HuH-7-SCID mice were inoculated directly into the tumor with
10.sup.4 PFU virus. Serum was collected on day 6 and 7 and titered
in Vero cells. .sup.eUnderlined values indicate a 2.5 or 3.5
log.sub.10PFU/ml reduction in titer in Vero cells or HuH-7 cells,
respectively, at indicated temp when compared to permissive temp
(35.degree. C.). .sup.xSmall plaque size at 35.degree. C.; small
plaques have a diameter of <1.0 mm compared to wild type plaque
diameter of 1.5-2.0 mm in Vero cells, or a diameter of <0.4 mm
compared to wild type plaque diameter of 0.75 to 1.0 mm in HuH-7
cells.
TABLE-US-00014 TABLE 14 Phenotypes of rDEN4 mutant viruses encoding
single mutations identified in 10 5-FU mutant viruses that are ts
in both Vero and HuH-7 cells. Replication in 7-day Replication in
HuH-7- rDEN4- Mean virus titer (log.sub.10PFU/ mice.sup.b SCID
mice.sup.d 5-FU Mutation ml) at indicated temp (.degree. C.) Mean
log.sub.10 Mean log.sub.10 mutant (nt Gene/ Vero cells HuH-7 cells
reduction from wt.sup.c reduction from wt.sup.c viruses position)
region 35 37 39 39 .DELTA..sup.a 35 37 38 39 .DELTA. n (-
log.sub.10PFU/g brain) n (log.sub.10PFU/ml serum) 239, 489 parent
7.6 6.8 5.6 3.3.sup.e 4.3 7.6 6.7 4.7 2.5 5.1 30 2.1 6 0.- 3 773
.sub. 4995.sup.f NS3 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0
<1.6 &- gt;4.8 6 2.9 473 parent 6.7 6.3 5.4 2.0 4.7 7.2 6.7
3.7 1.9 5.3 12 1.2 8 (+)0.3 4480 NS2B 6.7 6.3 6.0 5.7 1.0 7.6 7.2
6.0 5.2 2.4 6 0.7 .sub. 4995.sup.f NS3 5.7 4.9 3.6 <1.6 >4.1
6.4 5.7 4.0 <1.6 >- 4.8 6 2.9 759 parent 7.2 6.9 6.4 4.7 2.5
7.5 6.8 6.3 3.1 4.4 12 1.4 5 (+)0.4 .sub. 4995.sup.f NS3 5.7 4.9
3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >- 4.8 6 2.9 8020 NS5
7.1 6.6 6.7 5.9 1.2 7.4 7.1 6.1 5.4 2.0 6 0.5 816 parent 6.8 6.4
5.8 3.9 2.9 7.5 6.2 5.5 3.1 4.4 6 2.9 6 0.4 .sub. 4995.sup.f NS3
5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >- 4.8 6 2.9
7174 NS4B 6.9 7.1 6.9 6.1 0.8 7.5 7.2 7.1 5.6 1.9 6 0.6 938 parent
7.1 6.5 5.6 3.1 4.0 7.2 6.4 5.6 3.1 4.1 6 1.7 6 0.5 3442 NS1 5.1
3.6 4.3 2.1 3.0 5.9 4.9 3.9 <1.6 4.3 6 4.1 .sub. 4995.sup.f NS3
5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >- 4.8 6 2.9
10275 3 ' UTR 6.9 6.4 6.4 5.8 1.1 7.1 6.8 7.1 5.2 1.9 6 0.5 173
parent 7.0 6.1 3.2 2.9 4.1 7.0 3.2 3.0 2.1 4.9 18 2.2 6 1.1 7163
NS4B 7.8 7.7 7.6 7.2 0.6 8.0 7.7 7.5 7.4 0.6 6 (+)0.1 7849 NS5 7.0
6.7 3.7 2.1 4.9 7.7 5.5 3.6 2.4 5.3 6 3.1 8872 NS5 7.0 6.3 6.4 4.4
2.6 7.4 6.4 5.1 2.9 4.5 6 0.1 509 parent 6.2 5.8 5.5 3.4 2.8 6.5
6.1 4.5 <1.6 >4.9 6 1.9 6 1.5 4266 NS2B 5.9 6.1 6.1 5.2 0.7
6.7 6.1 5.7 5.3 1.4 6 1.0 8092 NS5 5.0.sup.x 4.6 4.6 <1.6
>3.4 5.6.sup.x 4.8 4.4 <1.6 >- 4.0 12 4.0 1033 parent 6.7
6.0 5.9 4.1 2.6 6.9 5.6 4.7 <1.6 >5.3 12 1.7 5 0.7- 4907 NS3
6.7 6.0 5.8 4.0 2.7 7.1 6.1 6.8 2.3 4.8 12 1.8 8730 NS5 7.0 6.7 6.6
6.7 0.3 7.6 7.0 7.2 6.6 1.0 12 0.6 9977 NS5 5.6 5.5 4.6 4.1 1.5 6.4
6.1 6.2 4.6 1.8 6 0.7 .sup.aReduction in mean virus titer
(log.sub.10PFU/ml) at 39.degree. C. compared to permissive
temperature (35.degree. C.). .sup.bGroups of 6 suckling mice were
inoculated i.c. with 10.sup.4 PFU of virus. Brains were removed 5
days later, homogenized, and titered in Vero cells.
.sup.cComparison of mean virus titers of mice inoculated with
mutant virus and concurrent DEN4 control. Bold denotes .gtoreq.50-
or .gtoreq.100- fold decrease in replication in suckling or
SCID-HuH-7 mice, respectively. .sup.dGroups of HuH-7-SCID mice were
inoculated directly into the tumor with 10.sup.4 PFU virus. Serum
was collected on day 6 and 7 and titered in Vero cells.
.sup.eUnderlined values indicate a 2.5 or 3.5 log.sub.10PFU/ml
reduction in titer in Vero cells or HuH-7 cells, respectively, at
indicated temp when compared to permissive temp (35.degree. C.).
.sup.fData represents the results from a single rDEN4-4995 virus.
.sup.xSmall plaque size at 35.degree. C.; small plaques have a
diameter of <1.0 mm compared to wild type plaque diameter of
1.5-2.0 mm in Vero cells, or a diameter of <0.4 mm compared to
wild type plaque diameter of 0.75 to 1.0 mm in HuH-7 cells.
TABLE-US-00015 TABLE 15 sp, ts and mouse attenuation phenotypes of
rDEN4 mutant viruses encoding single mutations identified in 3
HuH-7 cell-specific ts 5-FU mutant viruses. Replication in HuH-
Replication in 7- 7-SCID mice.sup.b day mice.sup.b Mean log.sub.10
rDEN4- Mean virus titer (log.sub.10PFU/ml) Mean log.sub.10
reduction 5-FU Mutation at indicated temp (.degree. C.) reduction
from from wt.sup.c mutant (nt Gene/ Vero cells HuH-7 cells wt.sup.c
(log.sub.10PFU/g (log.sub.10PFU/ml viruses position) region 35 37
39 39 .DELTA..sup.a 35 37 38 39 .DELTA. n b- rain).sub.10 n serum)
686 parent 7.0 6.7 6.7 6.4 0.6 7.3 6.8 6.4 2.2 5.1 12 3.8 6 1.2
3575 NS2A 6.9 6.9 7.1 7.0 0.1 7.9 6.8 6.9 4.9 3.0 12 2.3 nd.sup.e
4062 NS2A 6.8 6.6 6.3 4.7 2.1 6.9 6.8 7.0 <1.6 >5.3 12 2.2 nd
7163 NS4B 7.8 7.7 7.6 7.2 0.6 8.0 7.7 7.5 7.4 0.6 6 (+)0.1 nd 992
parent 7.3 7.1 6.8 5.9 1.4 7.4 6.9 5.0 <1.6 >5.8 6 2.7 7 1.3
5695 NS3 5.6 4.7 4.7 3.8 1.8 6.3 5.1 3.7 <1.6 >4.7 6 2.8 nd
7162 NS4B 7.2 7.3 6.6 6.8 0.4 7.4 7.3 7.3 6.6 0.8 8 0.3 nd 1175
parent 7.4 7.1 6.9 5.3 2.1 7.6 6.5 4.7 3.3 4.3 12 1.7 5 1.0 7153
NS4B 7.7 7.7 7.6 7.2 0.5 8.0 7.8 7.5 6.9 1.1 6 0.2 nd 10186 NS5 4.3
3.7 2.4 <1.6 >2.7 5.1 <1.6 <1.6 <1.6 >3.- 5 6 3.4
nd 10275 3' 6.9 6.4 6.4 5.8 1.1 7.1 6.8 7.1 5.2 1.9 6 0.5 nd UTR
.sup.aReduction in titer (log.sub.10PFU/ml) at 39.degree. C.
compared to permissive temperature (35.degree. C.). .sup.bGroups of
6 suckling mice were inoculated i.c. with 10.sup.4 PFU virus.
Brains were removed 5 days later, homogenized, and titered in Vero
cells. .sup.cDetermined by comparing mean viral titers of mice
inoculated with mutant virus and concurrent 2A-13 or rDEN4 wt
control. .sup.dUnderlined values indicate a 2.5 or 3.5
log.sub.10PFU/ml reduction in titer in Vero cells or HuH-7 cells,
respectively, at indicated temp when compared to permissive temp
(35.degree. C.).
TABLE-US-00016 TABLE 16 Temperature-sensitive (ts) and mouse brain
attenuation (att) phenotypes of additional rDEN4 viruses encoding
single 5-FU mutations. Gene/ Mean virus titer (log.sub.10PFU/
Replication in suckling mice.sup.b 5-FU region ml) at indicated
temp (.degree. C.) Mean virus titer .+-. Mean log.sub.10-unit
mutant containing Vero cells HuH-7 cells SE (log.sub.10PFU/g
reduction from virus Virus mutation 35 37 38 39 .DELTA..sup.a 35 37
38 39 .DELTA. n brain- ) value for wt.sup.c 695 rDEN4-40 5 ' UTR
7.4 7.2 6.7 6.2 1.2 7.6 7.5 7.1 5.8 1.8 nd.sup.f nd 718 rDEN4-4059
NS2A 7.0 6.7 6.4 6.2 0.8 7.7 7.1 7.0 6.6 1.1 nd nd 311 rDEN4-4896
NS3 7.0 6.1 5.9 4.2 2.8 6.9.sup.x 6.0 5.6 3.3 3.6 6 4.1 .+-. 0.4
2.0** 695 rDEN4-6106 NS3 6.8 6.3 5.9 3.9 2.9 7.1 6.0 5.2 3.4 3.7 nd
nd 631 rDEN4-6259 NS3 7.0 6.1 5.8 5.0 2.0 7.5 6.6 5.7 4.2 3.3 6 2.2
.+-. 0.2 3.9** .sub. 695.sup.e rDEN4-7546 NS4B 7.5 7.6 7.4 6.6 0.9
7.7 7.6 7.3 5.7 2.0 n- d nd 718 rDEN4-7630 NS5 7.0 6.9 6.9 6.4 0.6
7.4 7.4 7.2 6.8 0.6 6 5.0 .+-. 0.3 0.5 718 rDEN4-8281 NS5 6.4 6.6
6.7 5.4 1.0 7.6 7.6 7.0 5.1 2.5 6 5.0 .+-. 0.5 1.1 .sup.aReduction
in titer (log.sub.10PFU/ml) at 39.degree. C. compared to titer at
permissive temperature (35.degree. C.). .sup.b6 mice were
inoculated i.c. with 10.sup.4 PFU virus in 30 .mu.l inoculum.
Brains were removed 5 days later, homogenized, and titered on Vero
cells. Limit of detection is 2.0 log.sub.10PFU/g. .sup.cDetermined
by comparing mean viral titers of mice inoculated with sample virus
and wt rDEN4 control. .sup.dUnderlined values indicate a 2.5 or 3.5
log.sub.10PFU/ml reduction in titer in Vero cells or HuH-7 cells,
respectively, at indicated temperature when compared to permissive
temperature (35.degree. C.). .sup.eThe 7546 mutation is also
present in nine other 5-FU mutant viruses. .sup.xSmall plaque size
at 35.degree. C.; small plaques have a diameter of <0.4 mm
compared to wt plaque diameter of 0.75 to 1.0 mm in HuH-7 cells.
.sup.fnot determined **The att phenotype is defined as a reduction
of >1.5 log.sub.10PFU/g compared to wt virus.
TABLE-US-00017 TABLE 17 Growth of wt DEN-4 2A-13 in SCID mice
transplanted with HuH-7 cells..sup.a Virus titer Dose Mouse
log.sub.10PFU/ml serum log.sub.10PFU/g tissue (log.sub.10PFU/ml) #
day 3 day 5 Brain Liver Tumor 4 87 2.7 5.9 2.0 6.9 8.0 88 2.0 5.9
3.8 3.3 8.0 89 <1.7 6.2 2.7 3.6 8.0 90 1.7 3.5 3.2 3.0 7.0 5 84
<1.7 7.2 3.2 4.0 7.0 85 1.7 6.6 3.6 6.3 5.8 6 91 4.4 8.3 6.0 7.3
8.0 92 4.2 7.7 3.3 6.9 7.3 93 4.0 6.6 3.3 5.7 8.4 94 4.3 8.1 5.8
7.8 7.5 .sup.aSCID mice were injected i.p. with 10.sup.7 HuH-7
human hepatoma cells. Approximately 8 weeks later, groups of
tumor-bearing SCID-HuH-7 mice were inoculated with virus directly
into the tumor. Serum and tissues were collected on day 5,
processed, and titered in Vero cells.
TABLE-US-00018 TABLE 18 Combination of ts mutations, NS3 4995 and
NS5 7849, in rDEN4 results in an additive ts phenotype. Mean virus
titer (log.sub.10PFU/ml) Replication in suckling mice.sup.b at
indicated temp (.degree. C.) Mean virus Mean log.sub.10 Vero cells
HuH-7 cells titer .+-. SE reduction Virus 35 37 38 39 .DELTA..sup.a
35 37 38 39 .DELTA. (log.sub.10PFU/g brain) from wt.sup.c 2A-13 wt
7.1 7.1 6.9 6.8 0.3 7.4 7.3 6.7 6.4 1.0 6.9 .+-. 0.09 -- rDEN4 wt
7.0 6.8 6.6 6.4 0.6 7.5 7.3 6.7 6.4 1.1 6.5 .+-. 0.11 --
rDEN4.DELTA.30 7.0 6.7 6.2 6.2 0.8 7.5 7.0 6.5 5.1 2.4 5.9 .+-.
0.21 0.6 rDEN4-4995 5.7 4.9 3.6 <1.6.sup.d >4.1 6.4 5.7 4.0
<1.6 >4.8 3- .4 .+-. 0.10 3.1 rDEN4-7849 7.0 6.7 3.7 2.1 4.9
7.7 5.5 3.6 2.4 5.3 2.6 .+-. 0.29 3.9 rDEN4-4995-7849 5.9 2.8
<1.6 <1.6 >4.3 5.6 2.4 <1.6 <1.6 &g- t;4.0 2.3
.+-. 0.20 4.2 .sup.aReduction in titer (log.sub.10PFU/ml) at
39.degree. C. compared to titer at permissive temperature
(35.degree. C.). .sup.bGroups of 6 suckling mice were inoculated
i.c. with 10.sup.4 PFU virus. Brains were removed 5 days later,
homogenized, and titered in Vero cells. The limit of detection is
2.0 log.sub.10PFU/g. .sup.cDetermined by comparing mean viral
titers of mice inoculated with sample virus and rDEN4 wt control.
.sup.dUnderlined values indicate a 2.5 or 3.5 log.sub.10PFU/ml
reduction in titer in Vero cells or HuH-7 cells, respectively, at
ndicated temperature when compared to permissive temperature.
TABLE-US-00019 TABLE 19 The 5-FU mutations are compatible with the
.DELTA.30 mutation for replication in the brain of suckling mice.
Mean log.sub.10- No. of Mean virus titer .+-. SE unit reduction
Virus mice/group (log.sub.10PFU/g brain).sup.a from wt.sup.b rDEN4
12 6.0 .+-. 0.1 -- rDEN4.DELTA.30 12 5.3 .+-. 0.1 0.7
rDEN4-2650.sup.c 12 3.7 .+-. 0.2 2.3 rDEN4.DELTA.30-2650 12 3.9
.+-. 0.1 2.1 rDEN4-4995.sup.d 6 3.5 .+-. 0.2 2.5
rDEN4.DELTA.30-4995 6 2.7 .+-. 0.4 3.3 rDEN4-8092.sup.d 12 2.0 .+-.
0.1 4.0 rDEN4.DELTA.30-8092 6 3.2 .+-. 0.2 2.8 rDEN4-10634.sup.c 12
3.8 .+-. 0.1 2.2 rDEN4.DELTA.30-10634 12 3.6 .+-. 0.1 2.4
.sup.aGroups of 6 suckling mice were inoculated i.c. with 10.sup.4
PFU of virus. Brains were removed 5 days later, homogenized, and
titered in Vero cells. .sup.bComparison of mean virus titers of
mice inoculated with mutant virus and rDEN4 control. .sup.cMutation
restricts growth in both mouse brain and HuH-7-SCID mice.
.sup.dMutation restricts growth in mouse brain only. The 8092
mutation has not been tested in SCID-HuH7 mice.
TABLE-US-00020 TABLE 20 Temperature-sensitive and mouse brain
attenuation phenotypes of viruses bearing charge-cluster-to-alanine
mutations in the NS5 gene of DEN4. Replication in suckling
mice.sup.d Mean virus titer (log.sub.10PFU/ Mean ml at indicated
temperature (.degree. C.).sup.b titer .+-. SE Mean Changed # nt
Vero Cells HuH-7 Cells (log.sub.10PFU/g log reduction
Mutation.sup.a AA Pair changed 35 37 38 39 .DELTA..sup.c 35 37 38
39 .DELTA. n brain) from wt.sup.e wt (rDEN4) n/a 0 8.1 8.1 7.9 7.6
0.5 8.3 8.0 7.5 7.5 0.8 48 6.0 .+-. 0.16 -- deletion n/a 30 6.3 6.1
6.1 5.7 0.6 6.9 6.3 5.9 4.7 2.2 42 5.4 .+-. 0.22 0.6
(rDEN4.DELTA.30) 21-22 D R 4 7.2 6.8 6.7 6.1 1.1 7.6 7.1 7.0 4.7
2.9 6 5.0 .+-. 0.50 0.6 22-23 R K 4 7.0 7.8 6.9 3.7 3.3 7.6 7.6 6.5
<1.7 >5.9 6 2.6 .+-. 0.19 2.9 23-24 K E 3 6.7 6.6 6.0 6.5 0.2
7.1 7.3 5.6 <1.7 >5.4 18 4.7 .+-. 0.09 1.5 26-27 E E 3 7.8
7.6 6.8 4.0 3.8 8.4 8.2 7.3 4.9 3.5 6 5.7 .+-. 0.30 +0.1 46-47 K D
3 7.4 7.4 7.3 7.0 0.4 7.8 7.8 7.3 6.8 1.0 6 5.4 .+-. 0.42 0.5
157-158 E E 3 6.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 6 2.8
.+-. 0.31 2.7 200-201 K H 4 5.3 4.6 5.3 4.1 1.2 5.6 4.9 3.7 <1.7
>3.9 12 5.5 .+-. 0.45 0.8 246-247 R H 5 6.9 5.8 5.7 5.4 1.5 6.4
6.1 6.1 5.5 0.9 6 6.1 .+-. 0.17 +0.5 253-254 E K 4 7.1 6.9 6.8 7.0
0.1 7.9 7.5 7.6 6.8 1.1 6 6.2 .+-. 0.13 +0.6 356-357 K E 3 7.7 7.6
7.0 7.0 0.7 8.0 7.3 6.4 <1.7 >6.3 6 3.5 .+-. 0.58 2.0 387-388
K K 5 7.7 6.1 7.0 <1.7 >6.0 7.0 6.3 7.0 <1.7 >5.3 6 3.-
1 .+-. 0.33 2.4 388-389 K K 5 5.1 4.5 <1.7 <1.7 >3.4 6.1
5.0 <1.7 <1.7 >- 4.4 6 5.0 .+-. 0.23 1.4 396-397 R E 4 7.0
7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 18 5.4 .+-. 0.35 1.1
397-398 E E 2 7.0 7.1 7.0 3.0 4.0 8.0 7.6 7.0 <1.7 >6.3 6 6.0
.+-. 0.22 0.8 436-437 D K 4 4.5 3.3 3.0 2.0 2.5 5.7 4.5 <1.7
<1.7 >4.0 12 2.3 .+-. 0.14 3.9 500-501 R E 3 6.6 6.3 5.7 2.3
4.3 7.1 6.5 <1.7 <1.7 >5.4 6 6.9 .+-. 0.49 +0.7 520-521 E
E 3 5.6 4.7 4.3 <1.7 >3.9 6.7 5.7 <1.7 <1.7 >5.0 - 6
5.2 .+-. 0.48 0.2 523-524 D K 4 6.6 6.3 6.3 5.8 0.8 7.1 6.6 <1.7
<1.7 >5.4 6 4.2 .+-. 0.47 1.3 524-525 K K 5 7.1 6.9 6.9 6.6
0.5 7.8 7.4 7.0 5.3 2.5 6 3.4 .+-. 0.54 2.1 525-526 K D 4 7.8 7.1
7.6 6.8 1.0 7.9 7.7 8.0 6.9 1.0 6 3.7 .+-. 0.64 1.8 596-597 K D 3
4.6 4.0 2.6 <1.7 >2.9 5.7 4.9 4.0 <1.7 >4.0 6 5.- 9
.+-. 0.14 0.5 641-642 K E 4 7.3 6.9 6.9 5.2 2.1 7.8 7.5 7.2 6.9 0.9
6 4.7 .+-. 0.45 1.2 642-643 E R 3 6.8 6.1 4.0 3.3 3.5 7.5 7.1 6.6
3.0 4.5 12 2.6 .+-. 0.15 3.6 645-646 E K 4 6.3 5.3 5.9 3.1 3.2 6.4
5.8 5.5 4.5 1.9 6 5.4 .+-. 0.51 0.2 649-650 K E 3 6.9 6.8 6.9 6.3
0.6 7.1 7.3 7.5 7.0 0.1 12 6.4 .+-. 0.20 +0.2 654-655 D R 4 6.3 5.7
<1.7 <1.7 >4.6 7.0 7.1 4.6 <1.7 >5.3 - 12 1.8 .+-.
0.10 4.0 750-751 R E 3 7.1 7.1 6.9 5.7 1.4 7.8 6.9 6.5 5.6 2.2 6
6.0 .+-. 0.18 0.7 808-809 E D 3 4.6 4.1 <1.7 <1.7 >2.9 5.2
<1.7 <1.7 <1.7 - >3.5 6 1.8 .+-. 0.05 3.1 820-821 E D 2
6.3 6.3 5.6 <1.7 >4.6 6.9 6.0 5.7 <1.7 >5.2 6 5n- 5
.+-. 0.33 1.2 827-828 D K 4 6.9 6.3 6.3 5.9 1.0 7.5 6.9 5.0 <1.7
>5.8 6 3.6 .+-. 0.76 2.3 877-878 K E 3 7.6 7.3 7.0 7.0 0.6 7.9
7.9 7.3 5.8 2.1 12 4.4 .+-. 0.65 1.8 878-879 E E 3 7.6 7.3 7.3 7.1
0.5 8.1 8.1 7.9 6.6 1.5 12 2.4 .+-. 0.10 3.8 .sup.aPositions of the
amino acid pair mutated to an alanine pair; numbering starts at the
amino terminus of the NS5 protein. .sup.bUnderlined values indicate
a 2.5 or 3.5 log10 PFU/ml reduction in titer in Vero or HuH-7
cells, respectively, at the indicated temperatures when compared to
permissive temperature (35.degree. C.). .sup.cReduction in titer
(log10 PFU/ml) at 39.degree. C. compared to permissive temperature
(35.degree. C.). .sup.dGroups of six mice were inoculated i.c. with
4.0 log10 PFU virus in a 30 .mu.l inoculum. The brain was removed 5
days later, homogenized, and titered in Vero cells.
.sup.eDetermined by comparing mean viral titers in mice inoculated
with sample virus and concurrent wt controls (n = 6). The
attenuation phenotype is defined as a reduction of .gtoreq.1.5
log10 PFU/g compared to wt virus; reductions of .gtoreq.1.5 are
listed in boldface.
TABLE-US-00021 TABLE 21 SCID-HuH-7 attenuation phenotypes of
viruses bearing charge-cluster-to-alanine mutations in the NS5 gene
of DEN4. Replication in SCID-HuH-7 mice.sup.b AA Mean peak virus
titer .+-. SE Mean log Mutation.sup.a changed n (log.sub.10PFU/ml
serum) reduction from wt.sup.c wt na 21 5.4 .+-. 0.4 -- .DELTA.30
na 4 3.7 .+-. 0.6 2.5 23-24 KE 19 4.7 .+-. 0.5 1.3 157-158 EE 6 4.6
.+-. 0.6 1.3 200-201 KH 12 3.7 .+-. 0.2 2.6 356-357 KE 10 6.3 .+-.
0.7 (-) 1.1 396-397 RE 12 4.4 .+-. 1.3 1.2 397-398 EE 6 6.0 .+-.
0.5 (-) 0.1 436-437 DK 6 3.6 .+-. 0.2 2.6 500-501 RE 8 5.1 .+-. 0.4
1.1 523-524 DK 5 5.3 .+-. 0.7 0.6 750-751 RE 8 5.1 .+-. 0.4 1.1
808-809 ED 8 3.2 .+-. 0.4 3.0 827-828 DK 5 2.9 .+-. 0.2 1.6 878-879
EE 5 4.4 .+-. 0.7 1.5 .sup.aPositions of the amino acid pair
changed to a pair of alanines; numbering starts at the amino
terminus of the NS5 protein. .sup.bGroups of SCID-HuH-7 mice were
inoculated directly into the tumor with 10.sup.4 PFU virus. Serum
was collected on days 6 and 7 and titered in Vero cells.
.sup.cComparison ofmean virus titers ofmice inoculated with mutant
virus and concurrent DEN4 control. Bold denotes a .gtoreq.100-fold
decrease in replication. A (-) sign indicates an increase in
replication relative to wt.
TABLE-US-00022 TABLE 22 Combination of paired
charge-cluster-to-alanine mutations into double-pair mutant
viruses. Mutation Pair 1 Mutation Pair 2 Recovered 23-24 200-201
Yes 23-24 356-357 Yes 23-24 396-397 Yes 23-24 523-524 Yes 23-24
827-828 No 157-158 200-201 No 157-158 356-357 No 157-158 396-397 No
157-158 523-524 Yes 157-158 827-828 No 827-828 200-201 No 827-828
356-357 No 827-828 396-397 Yes 827-828 523-524 No
TABLE-US-00023 TABLE 23 Temperature-sensitive and mouse brain
attenuation phenotypes of double charge-cluster-to- alanine mutants
of the NS5 gene of rDEN4. Replication in suckling mice.sup.d Mean
virus titer (1og10 PFU/ml) at Mean virus indicated temperature
(.degree. C.).sup.b titer .+-. SE Mean log Charged # nt Vero Cells
HuH-7 cells (log.sub.10PFU/g reduction Mutation.sup.a AA Pair
changed 35 37 38 39 .DELTA..sup.c 35 37 38 39 .DELTA. n brain) from
wt.sup.e wt n/a 0 8.1 8.1 7.9 7.6 0.5 8.3 8.0 7.5 7.5 0.8 48 6.0
.+-. 0.16 -- .DELTA.30 n/a 30 6.3 6.1 6.1 5.7 0.6 6.9 6.3 5.9 4.7
2.2 42 5.4 .+-. 0.22 0.6 23-24 K E 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3
5.6 <1.7 >5.4 18 4.7 .+-. 0.09 1.5 200-201 K H 4 5.3 4.6 5.3
4.1 1.2 5.6 4.9 3.7 <1.7 >3.9 12 5.5 .+-. 0.45 0.8 23-24;
200-201 K E, K H 7 7.1 6.5 6.6 <1.7 >5.4 7.8 7.3 <1.7
<1.7 >6.1 6 5.8 .+-. 0.16 0.6 23-24 K E 3 6.7 6.6 6.0 6.5 0.2
7.1 7.3 5.6 <1.7 >5.4 18 4.7 .+-. 0.09 1.5 356-357 K E 3 7.7
7.6 7.0 7.0 0.7 8.0 7.3 6.4 <1.7 >6.3 6 3.5 .+-. 0.58 2.0
23-24; 356-357 K E, K E 6 23-24 K E 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3
5.6 <1.7 >5.4 18 4.7 .+-. 0.09 1.5 396-397 R E 4 7.0 7.3 6.5
5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 18 5.4 .+-. 0.35 1.1 23-24;
396-397 K E, R E 7 6.3 4.9 <1.7 <1.7 >4.6 7.1 6.0 5.6
<1.7 >5.4 6 3.7 .+-. 0.44 2.7 157-158 E E 3 6.5 7.2 5.1 5.1
1.4 7.6 7.4 5.9 <1.7 >5.9 6 2.8 .+-. 0.31 2.7 396-397 R E 4
7.0 7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 18 5.4 .+-. 0.35
1.1 157-158; 396-397 E E, R E 7 6 2.0 .+-. 0.12 4.8 157-158 E E 3
6.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 6 2.8 .+-. 0.31 2.7
523-524 D K 4 6.6 6.3 6.3 5.8 0.8 7.1 6.6 <1.7 <1.7 >5.4 6
4.2 .+-. 0.47 1.3 157-158; 523-524 E E, D K 7 5.6 3.9 <1.7
<1.7 >3.9 6.3 4.1 <1.7 <1.7 >4.6 396-397 R E 4 7.0
7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 6 4.8 .+-. 0.54 1.6
827-828 D K 4 6.9 6.3 6.3 5.9 1.0 7.5 6.9 5.0 <1.7 >5.8 6 3.6
.+-. 0.76 2.3 396-397; 827-828 R E, D K 8 7.0 6.5 6.0 <1.7 5.3
>6.7 5.7 <1.7 <1.7 >5.0 6 4.7 .+-. 0.10 1.2
.sup.aPositions of the amino acid pair mutated to an alanine pair;
numbering starts at the amino terminus of the NS5 protein.
.sup.bUnderlined values indicate a 2.5 or 3.5 log.sub.10PFU/ml
reduction in titer in Vero or HuH-7 cells respectively, at the
indicated temperatures when compared to permissive temperature
(35.degree. C.). .sup.cReduction in titer (log.sub.10PFU/ml) at
39.degree. C. compared to permissive temperature (35.degree. C.).
.sup.dGroups of six suckling mice were inoculated i.c. with 4.0
log.sub.10PFU virus in a 30 .mu.l inoculum. Brains were removed 5
days later, homogenized, and titered in Vero cells.
.sup.eDetermined by comparing mean viral titers in mice inoculated
with sample virus and concurrent wt controls (n = 6); reductions
.gtoreq.1.5 are listed in boldface.
TABLE-US-00024 TABLE 24 SCID-HuH-7 attenuation phenotypes of double
charge-cluster- to-alanine mutants of the NS5 gene of rDEN4.
Replication in SCID-HuH-7 mice.sup.b Mean peak virus Mean log
Charged titer .+-. SE reduction Mutation.sup.a AA Pair n
(log.sub.10PFU/ml serum) from wt.sup.c wt n/a 21 5.4 .+-. 0.4 --
.DELTA.30 n/a 4 3.7 .+-. 0.6 2.5 23-24 KE 19 4.7 .+-. 0.5 1.3
200-201 K H 12 3.7 .+-. 0.2 2.6 23-24; 200-201 K E, K H 13 3.4 .+-.
0.1 2.9 23-24 KE 19 4.7 .+-. 0.5 1.3 356-357 K E 10 6.3 .+-. 0.7
(+) 1.1 23-24; 356-357 K E, K E 4 3.6 .+-. 0.3 2.3 23-24 KE 19
4.7-.+-. 0.5 1.3 396-397 R E 12 4.4 .+-. 1.3 1.2 23-24; 396-397 K
E, R E 10 3.4 .+-. 0.5 3.3 157-158 EE 6 4.6 .+-. 0.6 1.3 396-397 R
E 12 4.4 .+-. 1.3 1.2 157-158; 396-397 E E, R E 6 2.2 .+-. 0.2 3.6
157-158 E E 6 4.6 .+-. 0.6 1.3 523-524 D K 5 5.3 .+-. 0.7 0.6
157-158; 523-524 E E, D K 3 5.1 .+-. 0.6 0.8 396-397 R E 12 4.4
.+-. 1.3 1.2 827-828 D K 5 2.9 .+-. 0.2 1.6 396-397; 827-828 R E, D
K 4 4.1 .+-. 0.7 0.4 .sup.aPositions of the amino acid pair mutated
to an alanine pair; numbering starts at the amino terminus of the
NS5 protein. .sup.bGroups of SCID-HuH-7 mice were inoculated
directly into the tumor with 10.sup.4 PFU of virus. Serum was
collected on days 6 and 7 and titered in Vero cells.
.sup.cComparison of mean virus titers of mice inoculated with
mutant virus and concurrent DEN4 control. Bold denotes a
.gtoreq.100-fold decrease in replication. A (+) sign indicates an
increase in replication relative to wt.
TABLE-US-00025 TABLE 25 Phenotypes (temperature sensitivity, plaque
size and replication in mouse brain and SCID-HuH-7 mice) of wt DEN4
and viruses containing the .DELTA.30 and 7129 mutations.
Replication in suckling Replication in Mean virus titer mouse
brain.sup.c SCID-HuH-7 mice.sup.e (log.sub.10 PFU/ml) at indicated
Mean virus Mean peak temperature (.degree. C.) titer .+-. SE Mean
log virus titer .+-. SE Mean log VERO HUH7 C6/36 (log.sub.10PFU/g
reduction (log.sub.10PFU/ml reduction- Virus ID Mutation.sup.a 35
39 .DELTA..sup.b 35 39 .DELTA. 32 n brain) from wt.sup.d n
serum).sup.f from wt.sup.d 1-TD- 1A wt 7.3 6.8 0.5 8 6.8 1.2 8.3 36
6.1 .+-. 0.21 -- 21 5.4 .+-. 0.4 -- p4.DELTA.30 .DELTA.30 6.6 6.5
0.1 7.4 6.4 1.0 42 5.4 .+-. 0.22 0.6 4 3.7 .+-. 0.6 2.5 5-1A1
C7129U 6.7 6.5 0.2 7.5 6 1.5 7.6* 6 6.2 .+-. 0.30 0.0 rDEN4-7129-1A
C7129U 7.3 7.0 0.3 7.6 6.3 1.3 7.5* 6 7.2 .+-. 0.12 (-)0.4 4 5.4
.+-. 0.8 (-)0.8 rDEN4.DELTA.30-7129 C7129U + 7.0 7.1* .DELTA.30
.sup.aPosition and identity of the mutated nucleotides.
.sup.bReduction in titer (log.sub.10PFU/ml) at 39.degree. C.
compared to permissive temperature (35.degree. C.). .sup.cGroups of
six suckling mice were inoculated i.c. with 4.0 log.sub.10PFU virus
in a 30 .mu.l inoculum. The brain was removed 5 days later,
homogenized, and titered in Vero cells. .sup.dDetermined by
comparing mean viral titers in mice inoculated with sample virus
and concurrent wt controls (n = 6). The attenuation phenotype is
defined as a .gtoreq.50- or .gtoreq.100-fold decrease in
replication in suckling or SCID-HuH-7 mice, respectively. A (-)
sign indicates an increase in replication relative to the wt
control. .sup.eGroups of SCID-HuH-7 mice were inoculated directly
into the tumor with 10.sup.4 PFU virus. Serum was collected on days
6 and 7 and titered in Vero cells. *Small plaque size.
TABLE-US-00026 TABLE 26 The 5-fluorouracil 5-1A1 small plaque
mutant demonstrates a restriction of midgut infection following
oral infection of Aedes aegytpi mosquitoes. Virus Dose ingested No.
mosquitoes Midgut-only Disseminated Total no. tested
(log.sub.10PFU).sup.a tested infection.sup.b infection.sup.c
infect- ed.sup.d,e wtDEN4 4.5 19 1 (5%) 17 (89%) 18 (95%) (2A-13)
3.5 26 9 (35%) 7 (27%) 16 (62%) 2.5 28 1 (4%) 0 1 (4%) OID.sub.50 =
3.9 OID.sub.50 = 3.3 5-1A1 3.5 34 4 (12%) 2 (6%) 6 (18%) 2.5 9 0 1
(11%) 1 (11%) 1.5 23 0 0 0 OID.sub.50 .gtoreq. 3.9 .sup.aAmount of
virus ingested, assuming a 2 .mu.l bloodmeal. .sup.bNumber
(percentage) of mosquitoes with detectable dengue virus antigen in
midgut tissue, but no detectable dengue virus antigen in head;
mosquitoes were assayed 21 days post-feed, and dengue virus antigen
was identified by IFA. .sup.cNumber (percentage) of mosquitoes with
detectable dengue virus antigen in both midgut and head tissue.
.sup.dTotal number (percentage) of mosquitoes with detectable
dengue virus antigen. .sup.eThe proportion of total infections
caused by wild type DEN4 was significantly higher than the
proportion caused by 5-1A1 (logistic regression, N = 426, P <
0.0001). There were too few disseminated infection caused by 5-1A1
to permit statistical analysis.
TABLE-US-00027 TABLE 27 The 5-fluorouracil 5-1A1 small plaque
mutant demonstrates a restriction of infection following
intrathoracic inoculation of Toxorhynchites splendens mosquitoes.
Virus Dose ingested No. mosquitoes tested (log.sub.10PFU).sup.a
tested No (%) infected.sup.c wtDEN4 4.0 5 5 (100) (2A-13) 3.0 4 4
(100) 2.0 4 1 (25) MID.sub.50 = 2.3 log.sub.10PFU 5-1A1 3.0 9 0 2.0
7 1 (14) 1.0 7 0 MID.sub.50 > 3.0 log.sub.10PFU .sup.aAmount of
virus inoculated in a 0.22 .mu.l inoculum. .sup.bNumber
(percentage) of mosquitoes with detectable dengue virus antigen in
head tissue; mosquitoes were assayed 14 days post-inoculation, and
dengue virus antigen was identified by IFA. .sup.cThe proportion of
infections caused by wild type DEN4 was significantly higher than
the proportion caused by 5-1A1 (logistic regression, N = 36, P <
0.01).
TABLE-US-00028 TABLE 28 Mutagenesis primers for the deletion or
swap of sequences in DEN4 showing conserved differences from
tick-borne flaviviruses. DEN4 nucleotides.sup.1 Type of
mutation.sup.2 Mutagenesis Primer.sup.3 SEQ ID NO 10508-10530
.DELTA. CTGGTGGAAGCCCAACACAAAAAC 64 10508-10530 swap
CTGGTGGAAGGAAGAGAGAAATTGGCAACTCCCCAACACAAAAAC 65 10535-10544
.DELTA. AGACCCCCCCAAGCATATTGAC 66 10535-10544 swap
AGACCCCCCCAATATTTCCTCCTCCTATAGCATATTGAC 67 10541-10544 .DELTA.
CCCAACACAAAGCATATTGAC 68 .sup.1Nucleotides numbered 5' to 3', in
the opposite direction from FIG. 5.3 .sup.2.DELTA.: deletion of
specified DEN4 nucleotides; swap: exhange of specified DEN4
nucleotides with homologous sequence from Langat .sup.3no swap
mutation was made for nucleotides 10541-10544
TABLE-US-00029 TABLE 29 Virus titer and plaque size of 3' UTR
mutant viruses in Vero and C6/36 cells. Vero C6/36 Titer
(log.sub.10 Plaque Titer (log.sub.10 Plaque Virus PFU/ml)
size.sup.1 PFU/ml) size rDEN4.DELTA.10508-10530 8.1 wt 7.5 wt
rDEN4swap10508-10530 5.4 sp 6.6 wt rDEN4.DELTA.10535-10544 5.8 wt
7.0 sp rDEN4swap10535-10544 7.0 wt 7.3 wt rDEN4.DELTA.10541-10544
6.4 wt >7.0 wt .sup.1Plaque size is designated as equivalent to
wild type (wt) or .gtoreq.50% of wild type (sp) on the designated
cell type.
TABLE-US-00030 TABLE 30 Infectivity of wt DEN4 and 3' UTR mutants
for Toxorhynchites splendens via intrathoracic inoculation. No.
Dose mosquitoes % MID.sub.50 Virus (log.sub.10PFU).sup.a tested
Infected.sup.b (log.sub.10 PFU) rDEN4 wt 3.3 6 83 2.3 2.3 7 57 1.3
6 0 0.3 6 0 rDEN4.DELTA.10508- 4.4 8 0 10530 3.4 9 11 2.4 4 0
.sup.aAmount of virus inoculated in a 0.22 .mu.l inoculum.
.sup.bPercentage of mosquitoes with detectable dengue virus antigen
in head tissue; mosquitoes were assayed 14 days post-inoculation,
and dengue virus antigen was identified by IFA
TABLE-US-00031 TABLE 31 Infectivity of 3' UTR swap mutant viruses
for Aedes aegypti fed on an infectious bloodmeal. Dose No. ingested
Mosquitoes Total No. Disseminated Virus Tested
(log.sub.10PFU).sup.a Tested Infected.sup.b,c Infections.sup.- c,d
rDEN4 3.8 18 11 (61%) 4 (22%) 2.8 15 5 (33%) 1 (6%) 1.8 15 0 0
OID.sub.50 = 3.4 OID.sub.50 = .gtoreq.4.2 rDEN4swap 3.8 25 5 (20%)
2 (8%) 10535-10544 2.8 25 0 0 1.8 20 0 0 OID.sub.50 = .gtoreq.4.2
.sup.aAmount of virus ingested, assuming a 2 .mu.l bloodmeal.
.sup.bNumber (%) of mosquitoes with detectable dengue virus antigen
in the midgut tissue; mosquitoes were assayed either 14 d post-feed
and dengue yins antigen was identified by IFA. .sup.cAt a dose of
3.8 log.sub.10PFU, rDEN4swap 10535-10544 infected significantly
fewer mosquitoes at the midgut than wt rDEN4 (Fisher's exact test,
N = 43, P < 0.01), although disseminated infections were not
significantly different (Fisher's exact test, N = 43, P = 0.38).
.sup.dNumber (%) of mosquitoes with detectable dengue virus antigen
in the head tissue.
TABLE-US-00032 TABLE 32 Putative Vero cell adaptation mutations
derived from the set of 5-FU mutant viruses and other DEN4 viruses
passaged in Vero cells. Other DEN viruses 5-FU mutant viruses
passaged in Vero cells Nucleotide Gene/region Nucleotide Amino acid
No. of viruses Nucleotide Amino acid position (a.a. #).sup.b change
change with the mutation Virus change change 1455 E (452) G > U
val > phe 5 .sub. 2280.sup.1,2,3 E (727) U > C phe > leu 2
.sub. 4891.sup.2,3 NS3 (1597) U > C ile > thr 2 .sub.
4995.sup.1,2 NS3 (1599) U > C ser > pro 8 7153 NS4B (2351) U
> C val > ala 3 2A.DELTA.30 U > C val > ala 7162 NS4B
(2354) U > C leu > ser 4 2A-1 U > C leu > ser 7163 NS4B
(2354) A > U or C leu > phe 7 rDEN4.DELTA.30 A > U leu
> phe 2A-13-1A1 A > U leu > phe .sub. 7182.sup.1,2,3 NS4B
(2361) G > A gly > ser 2 7546 NS4B (2482) C > U ala >
val 10 76303 NS5 (2510) A > G lys > arg 1 814669 A > G lys
> arg 10275 3 ' UTR A > U n/a.sup.c 6 10279 3 ' UTR A > C
n/a 4 .sup.aConservation with DEN1, DEN2, or DEN3 is designated by
superscript Lack of conservation is designated by no superscript.
.sup.bAmino acid position in DEN4 polyprotein beginning with the
methionine residue of the C protein (nt 102-104) as residue #1.
.sup.cnot applicable
TABLE-US-00033 TABLE 33 Sequence analysis of rDEN2/4.DELTA.30 clone
27(p4)-2-2A2. Mutation Nucleotide Gene Nucleotide Amino acid 743 M
anchor G > A Gly > Glu 1493 E C > U Ser > Phe 7544*
NS4B C > U Ala > Val *Same as DEN4 nucleotide position
7546
TABLE-US-00034 TABLE 34 Sequence analysis of rDEN2/4.DELTA.30 clone
27(p3)-2-1A1. Mutation Nucleotide Gene Nucleotide Amino acid 1345 E
U > C Tyr > His 4885* NS3 G > A Glu > Lys 8297 NS5 G
> A Arg > Lys *Codon adjacent to 5-FU mutation 4891
TABLE-US-00035 TABLE 35 Recombinant virus rDEN2/4.DELTA.30 bearing
Vero adaptation mutations can be recovery and titered on Vero
cells. Virus titer in Virus titer indicated cell line.sup.1
following recovery (log.sub.10PFU/ml) in Vero cells Virus C6/36
Vero (log.sub.10PFU/ml) rDEN2/4.DELTA.30 wt 5.2 1.7 <0.7
rDEN2/4.DELTA.30-7153 5.4 5.2 <0.7 rDEN2/4.DELTA.30-7162 5.4 5.3
nd.sup.2 rDEN2/4.DELTA.30-7182 4.7 4.9 2.3 rDEN2/4.DELTA.30-7630
5.3 4.8 1.3 rDEN2/4.DELTA.30-7153-7163 5.1 4.7 nd
rDEN2/4.DELTA.30-7153-7182 4.1 3.2 nd rDEN2/4.DELTA.30-7546-7630
5.2 5.2 nd .sup.1Virus recovered following transfection of C6/36
mosquito cells was terminally diluted once in C6/36 cells and
titered simultaneously in C6/36 cells and Vero cells. .sup.2not
determined
TABLE-US-00036 TABLE 36 Putative Vero cell adaptation mutations of
dengue type 4 virus and the corresponding wildtype amino acid
residue in other dengue viruses. Amino acid in indicated Amino acid
Mutant wt dengue virus.sup.b Mutation position.sup.a residue DEN4
DEN1 DEN2 DEN3 1455 452 F V I A A 2280 727 L F.sup.c F F F 4891
1597 T I V I I 4995 1632 P S S S N 7129 2343 L P P P P 7153 2351 A
V F F L 7162 2354 S L V V V 7163 2354 F L V V V 7182 2361 S G G G G
7546 2482 V A L T V 7630 2510 R K S S K .sup.aAmino acid position
is given for the polyprotein of DEN4 .sup.bDEN4 = rDEN4
(GenBankAF326825); DEN1 = Western pacific (GenBank DVU88535); DEN2
= New Guinea C (GenBankAF038403); DEN3 = H87 (GenBank M93130)
.sup.cUnderlined nucleotides are shared between DEN4 and one or
more additional DEN types.
TABLE-US-00037 TABLE 37 Mutations known to attenuate dengue type 4
virus and the corresponding wildtype amino acid residue in other
dengue virus. Amino acid Mutant Amino acid in indicated wt dengue
virus.sup.b Mutation position.sup.a residue DEN4 DEN1 DEN2 DEN3
5-FU mutations 2650 850 S N.sup.d N N N 3442 1114 G E E E E 3540
1147 K E E E E 3575 1158 I M L A M 3771 1224 G R R K R 4062 1321 A
T L A T 4306 1402 S N E D D 4891 1597 T I V I I 4896 1599 S A A A A
4907 1602 F L L L L 4995 1632 P S S S N 5097 1666 N D D D D 5695
1865 G D D D D 6259 2053 A V V V V .sub. 7129.sup.c 2343 L P P P P
7849 2583 I N K N K 8092 2664 G E Q Q Q 10186 3362 T I I I I 10634
3 ' UTR -- -- -- -- -- Charge-cluster-to- 22, 23 2509, 2510 AA RK
KS KS RK alanine mutations 23, 24 2510, 2511 AA KE SE SE KE 157,
158 2644, 2645 AA EE EE EA EE 200, 201 2687, 2688 AA KH KH KY KH
356, 357 2843, 2844 AA KE KE KE KE 387, 388 2874, 2875 AA KK RN KK
RN 436, 437 2923, 2924 AA DK HR DK DK 524, 525 3011, 3012 AA KK KI
KK KI 525, 526 3012, 3013 AA KD IP KE IP 642, 643 3129, 3130 AA ER
ER IA KK 654, 655 3141, 3142 AA DR ER ER ER 808, 809 3295, 3296 AA
ED ED ED ED 827, 828 3314, 3315 AA DK DK DK DK 877, 878 3364, 3365
AA KE NE NE NE 878, 879 3365, 3366 AA EE EN EE EE .sup.aAmino acid
position is given for the polyprotein of DEN4 .sup.bDEN4 = rDEN4
(GenBank AF326825); DEN1 = Western pacific (GenBank U88535); DEN2 =
New Guinea C (GenBank AF038403); DEN3 = H87 (GenBank M93130)
.sup.cThis mutation results in decreased replication of DEN4 in
mosquitoes. .sup.dUnderlined nucleotides are shared between DEN4
and one or more additional DEN types.
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, and appendices, as well as patents, applications,
and publications, referred to above, are hereby incorporated by
reference.
SEQUENCE LISTINGS
1
701900PRTDengue 4 virus 1Gly Thr Gly Thr Thr Gly Glu Thr Leu Gly
Glu Lys Trp Lys Arg Gln 1 5 10 15 Leu Asn Ser Leu Asp Arg Lys Glu
Phe Glu Glu Tyr Lys Arg Ser Gly 20 25 30 Ile Leu Glu Val Asp Arg
Thr Glu Ala Lys Ser Ala Leu Lys Asp Gly 35 40 45 Ser Lys Ile Lys
His Ala Val Ser Arg Gly Ser Ser Lys Ile Arg Trp 50 55 60 Ile Val
Glu Arg Gly Met Val Lys Pro Lys Gly Lys Val Val Asp Leu65 70 75 80
Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Met Ala Thr Leu Lys Asn 85
90 95 Val Thr Glu Val Lys Gly Tyr Thr Lys Gly Gly Pro Gly His Glu
Glu 100 105 110 Pro Ile Pro Met Ala Thr Tyr Gly Trp Asn Leu Val Lys
Leu His Ser 115 120 125 Gly Val Asp Val Phe Tyr Lys Pro Thr Glu Gln
Val Asp Thr Leu Leu 130 135 140 Cys Asp Ile Gly Glu Ser Ser Ser Asn
Pro Thr Ile Glu Glu Gly Arg145 150 155 160 Thr Leu Arg Val Leu Lys
Met Val Glu Pro Trp Leu Ser Ser Lys Pro 165 170 175 Glu Phe Cys Ile
Lys Val Leu Asn Pro Tyr Met Pro Thr Val Ile Glu 180 185 190 Glu Leu
Glu Lys Leu Gln Arg Lys His Gly Gly Asn Leu Val Arg Cys 195 200 205
Pro Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Gly Ala 210
215 220 Ser Gly Asn Ile Val Ser Ser Val Asn Thr Thr Ser Lys Met Leu
Leu225 230 235 240 Asn Arg Phe Thr Thr Arg His Arg Lys Pro Thr Tyr
Glu Lys Asp Val 245 250 255 Asp Leu Gly Ala Gly Thr Arg Ser Val Ser
Thr Glu Thr Glu Lys Pro 260 265 270 Asp Met Thr Ile Ile Gly Arg Arg
Leu Gln Arg Leu Gln Glu Glu His 275 280 285 Lys Glu Thr Trp His Tyr
Asp Gln Glu Asn Pro Tyr Arg Thr Trp Ala 290 295 300 Tyr His Gly Ser
Tyr Glu Ala Pro Ser Thr Gly Ser Ala Ser Ser Met305 310 315 320 Val
Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp Val Ile Pro 325 330
335 Met Val Thr Gln Leu Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln
340 345 350 Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro Gln Pro
Lys Pro 355 360 365 Gly Thr Arg Met Val Met Thr Thr Thr Ala Asn Trp
Leu Trp Ala Leu 370 375 380 Leu Gly Lys Lys Lys Asn Pro Arg Leu Cys
Thr Arg Glu Glu Phe Ile385 390 395 400 Ser Lys Val Arg Ser Asn Ala
Ala Ile Gly Ala Val Phe Gln Glu Glu 405 410 415 Gln Gly Trp Thr Ser
Ala Ser Glu Ala Val Asn Asp Ser Arg Phe Trp 420 425 430 Glu Leu Val
Asp Lys Glu Arg Ala Leu His Gln Glu Gly Lys Cys Glu 435 440 445 Ser
Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu 450 455
460 Phe Gly Arg Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu
Gly465 470 475 480 Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu
Asn Glu Asp His 485 490 495 Trp Phe Gly Arg Glu Asn Ser Trp Ser Gly
Val Glu Gly Glu Gly Leu 500 505 510 His Arg Leu Gly Tyr Ile Leu Glu
Glu Ile Asp Lys Lys Asp Gly Asp 515 520 525 Leu Met Tyr Ala Asp Asp
Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu 530 535 540 Asp Asp Leu Gln
Asn Glu Glu Leu Ile Thr Glu Gln Met Ala Pro His545 550 555 560 His
Lys Ile Leu Ala Lys Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys 565 570
575 Val Val Lys Val Leu Arg Pro Thr Pro Arg Gly Ala Val Met Asp Ile
580 585 590 Ile Ser Arg Lys Asp Gln Arg Gly Ser Gly Gln Val Gly Thr
Tyr Gly 595 600 605 Leu Asn Thr Phe Thr Asn Met Glu Val Gln Leu Ile
Arg Gln Met Glu 610 615 620 Ala Glu Gly Val Ile Thr Gln Asp Asp Met
Gln Asn Pro Lys Gly Leu625 630 635 640 Lys Glu Arg Val Glu Lys Trp
Leu Lys Glu Cys Gly Val Asp Arg Leu 645 650 655 Lys Arg Met Ala Ile
Ser Gly Asp Asp Cys Val Val Lys Pro Leu Asp 660 665 670 Glu Arg Phe
Gly Thr Ser Leu Leu Phe Leu Asn Asp Met Gly Lys Val 675 680 685 Arg
Lys Asp Ile Pro Gln Trp Glu Pro Ser Lys Gly Trp Lys Asn Trp 690 695
700 Gln Glu Val Pro Phe Cys Ser His His Phe His Lys Ile Phe Met
Lys705 710 715 720 Asp Gly Arg Ser Leu Val Val Pro Cys Arg Asn Gln
Asp Glu Leu Ile 725 730 735 Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly
Trp Ser Leu Arg Glu Thr 740 745 750 Ala Cys Leu Gly Lys Ala Tyr Ala
Gln Met Trp Ser Leu Met Tyr Phe 755 760 765 His Arg Arg Asp Leu Arg
Leu Ala Ser Met Ala Ile Cys Ser Ala Val 770 775 780 Pro Thr Glu Trp
Phe Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala785 790 795 800 His
His Gln Trp Met Thr Thr Glu Asp Met Leu Lys Val Trp Asn Arg 805 810
815 Val Trp Ile Glu Asp Asn Pro Asn Met Thr Asp Lys Thr Pro Val His
820 825 830 Ser Trp Glu Asp Ile Pro Tyr Leu Gly Lys Arg Glu Asp Leu
Trp Cys 835 840 845 Gly Ser Leu Ile Gly Leu Ser Ser Arg Ala Thr Trp
Ala Lys Asn Ile 850 855 860 His Thr Ala Ile Thr Gln Val Arg Asn Leu
Ile Gly Lys Glu Glu Tyr865 870 875 880 Val Asp Tyr Met Pro Val Met
Lys Arg Tyr Ser Ala Pro Ser Glu Ser 885 890 895 Glu Gly Val Leu 900
2233DNADengue 4 virus 2gactagcggt tagaggagac ccctcccatc actgataaaa
cgcagcaaaa gggggcccga 60agccaggagg aagctgtact cctggtggaa ggactagagg
ttagaggaga cccccccaac 120acaaaaacag catattgacg ctgggaaaga
ccagagatcc tgctgtctct gcaacatcaa 180tccaggcaca gagcgccgca
agatggattg gtgttgttga tccaacaggt tct 2333228DNADengue 1 virus
3gactagtggt tagaggagac ccctcccaag acacaacgca gcagcggggc ccaacaccag
60gggaagctgt accctggtgg taaggactag aggttagagg agaccccccg cacaacaaca
120aacagcatat tgacgctggg agagaccaga gatcctgctg tctctacagc
atcattccag 180gcacagaacg ccaaaaaatg gaatggtgct gttgaatcaa caggttct
2284230DNADengue 2 virus 4gactagcggt tagaggagac ccctccctta
caaatcgcag caacaatggg ggcccaaggt 60gagatgaagc tgtagtctca ctggaaggac
tagaggttag aggagacccc cccaaaacaa 120aaaacagcat attgacgctg
ggaaagacca gagatcctgc tgtctcctca gcatcattcc 180aggcacagaa
cgccagaaaa tggaatggtg ctgttgaatc aacaggttct 2305227DNADengue 3
virus 5gactagtggt tagaggagac ccctcccatg acacaacgca gcagcggggc
ccgagcactg 60agggaagctg tacctccttg caaaggacta gaggttatag gagacccccc
gcaaacaaaa 120acagcatatt gacgctggga gagaccagag atcctgctgt
ctcctcagca tcattccagg 180cacagaacgc cagaaaatgg aatggtgctg
ttgaatcaac aggttct 2276227DNAWest Nile virus 6gactagaggt tagaggagac
cccgcgtaaa aaagtgcacg gcccaacttg gctgaagctg 60taagccaagg gaaggactag
aggttagagg agaccccgtg ccaaaaacac caaaagaaac 120agcatattga
cacctgggat agactagggg atcttctgct ctgcacaacc agccacacgg
180cacagtgcgc cgacataggt ggctggtggt gctagaacac aggatct
2277229DNAJapanese encephalitis virus 7gactagaggt tagaggagac
cccgtggaaa caacaacatg cggcccaagc cccctcgaag 60ctgtagagga ggtggaagga
ctagaggtta gaggagaccc cgcatttgca tcaaacagca 120tattgacacc
tgggaataga ctgggagatc ttctgctcta tctcaacatc agctactagg
180cacagagcgc cgaagtatgt acgtggtggt gaggaagaac acaggatct
2298241DNAYellow fever virus 8aacctggttt ctgggacctc ccaccccaga
gtaaaaagaa cggagcctcc gctaccaccc 60tcccacgtgg tggtagaaag acggggtcta
gaggttagag gagaccctcc agggaacaaa 120tagtgggacc atattgacgc
cagggaaaga ccggagtggt tctctgcttt tcctccagag 180gtctgtgagc
acagtttgct caagaataag cagacctttg gatgacaaac acaaaaccac 240t
2419249DNAPowassan virus 9aaacgaactt tgtgagacca aaaggcctcc
tggaaggctc accaggagtt aggccgttta 60ggagcccccg agcataactc gggaggaggg
aggaagaaaa ttggcaatct tcctcgggat 120ttttccgcct cctatactaa
atttccccca ggaaactggg ggggcggttc ttgttctccc 180tgagccacca
ccatccaggc acagatagcc tgacaaggag atggtgtgtg actcggaaaa 240acacccgct
24910250DNALouping ill virus 10tgcaagattt tgcgagaccc cccgccccat
gacaaggccg aacatggagc attaaaggga 60ggcccccgga agcatgcttc cgggaggagg
gaagagagaa attggcagct ctcttcaggg 120tttttcctcc tcctatacca
aatttccccc tcgacagagg gggggcggtt cttgttctcc 180ctgagccacc
atcacccaga cacagatagt ctgacaagga ggtgatgtgt gactcggaaa
240aacacccgct 25011250DNATick-borne encephalitis virus 11tgaaaaattt
tgtgagaccc cctgcatcat gataaggccg aacatggtgc atgaaagggg 60aggcccccgg
aagcacgctt ccgggaggag ggaagagaga aattggcagc tctcttcagg
120atttttcctc ctcctataca aaattccccc tcggtagagg gggggcggtt
cttgttctcc 180ctgagccacc atcacccaga cacaggtagt ctgacaagga
ggtgatgtgt gactcggaaa 240aacacccgct 25012247DNALangat virus
12tgtgaaactt tgtgagaccc cttgcgtcca gagaaggccg aactgggcgt tataaggagg
60cccccagggg gaaacccctg ggaggaggga agagagaaat tggcaactct cttcaggata
120tttcctcctc ctataccaaa ttccccctcg tcagaggggg ggcggttctt
gttctccctg 180agccaccatc acctagacac agatagtctg aaaaggaggt
gatgcgtgtc tcggaaaaac 240acccgct 247133387PRTDengue 4 virus strain
2A 13Met Asn Gln Arg Lys Lys Val Val Arg Pro Pro Phe Asn Met Leu
Lys 1 5 10 15 Arg Glu Arg Asn Arg Val Ser Thr Pro Gln Gly Leu Val
Lys Arg Phe 20 25 30 Ser Thr Gly Leu Phe Ser Gly Lys Gly Pro Leu
Arg Met Val Leu Ala 35 40 45 Phe Ile Thr Phe Leu Arg Val Leu Ser
Ile Pro Pro Thr Ala Gly Ile 50 55 60 Leu Lys Arg Trp Gly Gln Leu
Lys Lys Asn Lys Ala Ile Lys Ile Leu65 70 75 80 Ile Gly Phe Arg Lys
Glu Ile Gly Arg Met Leu Asn Ile Leu Asn Gly 85 90 95 Arg Lys Arg
Ser Thr Ile Thr Leu Leu Cys Leu Ile Pro Thr Val Met 100 105 110 Ala
Phe Ser Leu Ser Thr Arg Asp Gly Glu Pro Leu Met Ile Val Ala 115 120
125 Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly Ile
130 135 140 Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys
Glu Asp145 150 155 160 Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn
Thr Glu Pro Glu Asp 165 170 175 Ile Asp Cys Trp Cys Asn Leu Thr Ser
Thr Trp Val Met Tyr Gly Thr 180 185 190 Cys Thr Gln Ser Gly Glu Arg
Arg Arg Glu Lys Arg Ser Val Ala Leu 195 200 205 Thr Pro His Ser Gly
Met Gly Leu Glu Thr Arg Ala Glu Thr Trp Met 210 215 220 Ser Ser Glu
Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp Ile225 230 235 240
Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr Met 245
250 255 Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met
Met 260 265 270 Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val
Gly Asn Arg 275 280 285 Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp
Val Asp Leu Val Leu 290 295 300 Glu His Gly Gly Cys Val Thr Thr Met
Ala Gln Gly Lys Pro Thr Leu305 310 315 320 Asp Phe Glu Leu Thr Lys
Thr Thr Ala Lys Glu Val Ala Leu Leu Arg 325 330 335 Thr Tyr Cys Ile
Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr Arg 340 345 350 Cys Pro
Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln Gln 355 360 365
Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly Cys 370
375 380 Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser
Cys385 390 395 400 Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu
Asn Leu Glu Tyr 405 410 415 Thr Val Val Val Thr Val His Asn Gly Asp
Thr His Ala Val Gly Asn 420 425 430 Asp Thr Ser Asn His Gly Val Thr
Ala Met Ile Thr Pro Arg Ser Pro 435 440 445 Ser Val Glu Val Lys Leu
Pro Asp Tyr Gly Glu Leu Thr Leu Asp Cys 450 455 460 Glu Pro Arg Ser
Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys Met465 470 475 480 Lys
Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu Pro 485 490
495 Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn Tyr
500 505 510 Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg
Gln Asp 515 520 525 Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His
Ser Ala Leu Ala 530 535 540 Gly Ala Thr Glu Val Asp Ser Gly Asp Gly
Asn His Met Phe Ala Gly545 550 555 560 His Leu Lys Cys Lys Val Arg
Met Glu Lys Leu Arg Ile Lys Gly Met 565 570 575 Ser Tyr Thr Met Cys
Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala 580 585 590 Glu Thr Gln
His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala 595 600 605 Gly
Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu 610 615
620 Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn
Thr625 630 635 640 Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe
Gly Asp Ser Tyr 645 650 655 Ile Val Ile Gly Val Gly Asn Ser Ala Leu
Thr Leu His Trp Phe Arg 660 665 670 Lys Gly Ser Ser Ile Gly Lys Met
Phe Glu Ser Thr Tyr Arg Gly Ala 675 680 685 Lys Arg Met Ala Ile Leu
Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 690 695 700 Gly Gly Leu Phe
Thr Ser Leu Gly Lys Ala Val His Gln Val Phe Gly705 710 715 720 Ser
Val Tyr Thr Thr Met Phe Gly Gly Val Ser Trp Met Ile Arg Ile 725 730
735 Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr
740 745 750 Ser Met Ala Met Thr Cys Ile Ala Val Gly Gly Ile Thr Leu
Phe Leu 755 760 765 Gly Phe Thr Val Gln Ala Asp Met Gly Cys Val Val
Ser Trp Ser Gly 770 775 780 Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe
Val Val Asp Asn Val His785 790 795 800 Thr Trp Thr Glu Gln Tyr Lys
Phe Gln Pro Glu Ser Pro Ala Arg Leu 805 810 815 Ala Ser Ala Ile Leu
Asn Ala His Lys Asp Gly Val Cys Gly Ile Arg 820 825 830 Ser Thr Thr
Arg Leu Glu Asn Val Met Trp Lys Gln Ile Thr Asn Glu 835 840 845 Leu
Asn Tyr Val Leu Trp Glu Gly Gly His Asp Leu Thr Val Val Ala 850 855
860 Gly Asp Val Lys Gly Val Leu Thr Lys Gly Lys Arg Ala Leu Thr
Pro865 870 875 880 Pro Val Ser Asp Leu Lys Tyr Ser Trp Lys Thr Trp
Gly Lys Ala Lys 885 890 895 Ile Phe Thr Pro Glu Ala Arg Asn Ser Thr
Phe Leu Ile Asp Gly Pro 900
905 910 Asp Thr Ser Glu Cys Pro Asn Glu Arg Arg Ala Trp Asn Ser Leu
Glu 915 920 925 Val Glu Asp Tyr Gly Phe Gly Met Phe Thr Thr Asn Ile
Trp Met Lys 930 935 940 Phe Arg Glu Gly Ser Ser Glu Val Cys Asp His
Arg Leu Met Ser Ala945 950 955 960 Ala Ile Lys Asp Gln Lys Ala Val
His Ala Asp Met Gly Tyr Trp Ile 965 970 975 Glu Ser Ser Lys Asn Gln
Thr Trp Gln Ile Glu Lys Ala Ser Leu Ile 980 985 990 Glu Val Lys Thr
Cys Leu Trp Pro Lys Thr His Thr Leu Trp Ser Asn 995 1000 1005 Gly
Val Leu Glu Ser Gln Met Leu Ile Pro Lys Ser Tyr Ala Gly Pro 1010
1015 1020 Phe Ser Gln His Asn Tyr Arg Gln Gly Tyr Ala Thr Gln Thr
Val Gly1025 1030 1035 1040Pro Trp His Leu Gly Lys Leu Glu Ile Asp
Phe Gly Glu Cys Pro Gly 1045 1050 1055 Thr Thr Val Thr Ile Gln Glu
Asp Cys Asp His Arg Gly Pro Ser Leu 1060 1065 1070 Arg Thr Thr Thr
Ala Ser Gly Lys Leu Val Thr Gln Trp Cys Cys Arg 1075 1080 1085 Ser
Cys Thr Met Pro Pro Leu Arg Phe Leu Gly Glu Asp Gly Cys Trp 1090
1095 1100 Tyr Gly Met Glu Ile Arg Pro Leu Ser Glu Lys Glu Glu Asn
Met Val1105 1110 1115 1120Lys Ser Gln Val Thr Ala Gly Gln Gly Thr
Ser Glu Thr Phe Ser Met 1125 1130 1135 Gly Leu Leu Cys Leu Thr Leu
Phe Val Glu Glu Cys Leu Arg Arg Arg 1140 1145 1150 Val Thr Arg Lys
His Met Ile Leu Val Val Val Ile Thr Leu Cys Ala 1155 1160 1165 Ile
Ile Leu Gly Gly Leu Thr Trp Met Asp Leu Leu Arg Ala Leu Ile 1170
1175 1180 Met Leu Gly Asp Thr Met Ser Gly Arg Ile Gly Gly Gln Ile
His Leu1185 1190 1195 1200Ala Ile Met Ala Val Phe Lys Met Ser Pro
Gly Tyr Val Leu Gly Val 1205 1210 1215 Phe Leu Arg Lys Leu Thr Ser
Arg Glu Thr Ala Leu Met Val Ile Gly 1220 1225 1230 Met Ala Met Thr
Thr Val Leu Ser Ile Pro His Asp Leu Met Glu Leu 1235 1240 1245 Ile
Asp Gly Ile Ser Leu Gly Leu Ile Leu Leu Lys Ile Val Thr Gln 1250
1255 1260 Phe Asp Asn Thr Gln Val Gly Thr Leu Ala Leu Ser Leu Thr
Phe Ile1265 1270 1275 1280Arg Ser Thr Met Pro Leu Val Met Ala Trp
Arg Thr Ile Met Ala Val 1285 1290 1295 Leu Phe Val Val Thr Leu Ile
Pro Leu Cys Arg Thr Ser Cys Leu Gln 1300 1305 1310 Lys Gln Ser His
Trp Val Glu Ile Thr Ala Leu Ile Leu Gly Ala Gln 1315 1320 1325 Ala
Leu Pro Val Tyr Leu Met Thr Leu Met Lys Gly Ala Ser Arg Arg 1330
1335 1340 Ser Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Leu Val
Ser Leu1345 1350 1355 1360Leu Gly Ser Ala Leu Leu Lys Asn Asp Val
Pro Leu Ala Gly Pro Met 1365 1370 1375 Val Ala Gly Gly Leu Leu Leu
Ala Ala Tyr Val Met Ser Gly Ser Ser 1380 1385 1390 Ala Asp Leu Ser
Leu Glu Lys Ala Ala Asn Val Gln Trp Asp Glu Met 1395 1400 1405 Ala
Asp Ile Thr Gly Ser Ser Pro Ile Ile Glu Val Lys Gln Asp Glu 1410
1415 1420 Asp Gly Ser Phe Ser Ile Arg Asp Val Glu Glu Thr Asn Met
Ile Thr1425 1430 1435 1440Leu Leu Val Lys Leu Ala Leu Ile Thr Val
Ser Gly Leu Tyr Pro Leu 1445 1450 1455 Ala Ile Pro Val Thr Met Thr
Leu Trp Tyr Met Trp Gln Val Lys Thr 1460 1465 1470 Gln Arg Ser Gly
Ala Leu Trp Asp Val Pro Ser Pro Ala Ala Thr Lys 1475 1480 1485 Lys
Ala Ala Leu Ser Glu Gly Val Tyr Arg Ile Met Gln Arg Gly Leu 1490
1495 1500 Phe Gly Lys Thr Gln Val Gly Val Gly Ile His Met Glu Gly
Val Phe1505 1510 1515 1520His Thr Met Trp His Val Thr Arg Gly Ser
Val Ile Cys His Glu Thr 1525 1530 1535 Gly Arg Leu Glu Pro Ser Trp
Ala Asp Val Arg Asn Asp Met Ile Ser 1540 1545 1550 Tyr Gly Gly Gly
Trp Arg Leu Gly Asp Lys Trp Asp Lys Glu Glu Asp 1555 1560 1565 Val
Gln Val Leu Ala Ile Glu Pro Gly Lys Asn Pro Lys His Val Gln 1570
1575 1580 Thr Lys Pro Gly Leu Phe Lys Thr Leu Thr Gly Glu Ile Gly
Ala Val1585 1590 1595 1600Thr Leu Asp Phe Lys Pro Gly Thr Ser Gly
Ser Pro Ile Ile Asn Arg 1605 1610 1615 Lys Gly Lys Val Ile Gly Leu
Tyr Gly Asn Gly Val Val Thr Lys Ser 1620 1625 1630 Gly Asp Tyr Val
Ser Ala Ile Thr Gln Ala Glu Arg Ile Gly Glu Pro 1635 1640 1645 Asp
Tyr Glu Val Asp Glu Asp Ile Phe Arg Lys Lys Arg Leu Thr Ile 1650
1655 1660 Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Ile Leu
Pro Ser1665 1670 1675 1680Ile Val Arg Glu Ala Leu Lys Arg Arg Leu
Arg Thr Leu Ile Leu Ala 1685 1690 1695 Pro 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 Val Lys Ser Glu His Thr Gly Arg 1715 1720 1725 Glu
Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Thr Arg Leu Leu 1730
1735 1740 Ser Ser Thr Arg Val Pro Asn Tyr Asn Leu Ile Val Met Asp
Glu Ala1745 1750 1755 1760His Phe Thr Asp Pro Ser Ser Val Ala Ala
Arg Gly Tyr Ile Ser Thr 1765 1770 1775 Arg Val Glu Met Gly Glu Ala
Ala Ala Ile Phe Met Thr Ala Thr Pro 1780 1785 1790 Pro Gly Ala Thr
Asp Pro Phe Pro Gln Ser Asn Ser Pro Ile Glu Asp 1795 1800 1805 Ile
Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Thr Gly Phe Asp Trp 1810
1815 1820 Ile Thr Asp Tyr Gln Gly Lys Thr Val Trp Phe Val Pro Ser
Ile Lys1825 1830 1835 1840Ala Gly Asn Asp Ile Ala Asn Cys Leu Arg
Lys Ser Gly Lys Lys Val 1845 1850 1855 Ile Gln Leu Ser Arg Lys Thr
Phe Asp Thr Glu Tyr Pro Lys Thr Lys 1860 1865 1870 Leu Thr Asp Trp
Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly 1875 1880 1885 Ala
Asn Phe Arg Ala Gly Arg Val Ile Asp Pro Arg Arg Cys Leu Lys 1890
1895 1900 Pro Val Ile Leu Pro Asp Gly Pro Glu Arg Val Ile Leu Ala
Gly Pro1905 1910 1915 1920Ile Pro Val Thr Pro Ala Ser Ala Ala Gln
Arg Arg Gly Arg Ile Gly 1925 1930 1935 Arg Asn Pro Ala Gln Glu Asp
Asp Gln Tyr Val Phe Ser Gly Asp Pro 1940 1945 1950 Leu Lys Asn Asp
Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu 1955 1960 1965 Leu
Asp Asn Ile Tyr Thr Pro Glu Gly Ile Ile Pro Thr Leu Phe Gly 1970
1975 1980 Pro Glu Arg Glu Lys Thr Gln Ala Ile Asp Gly Glu Phe Arg
Leu Arg1985 1990 1995 2000Gly Glu Gln Arg Lys Thr Phe Val Glu Leu
Met Arg Arg Gly Asp Leu 2005 2010 2015 Pro Val Trp Leu Ser Tyr Lys
Val Ala Ser Ala Gly Ile Ser Tyr Lys 2020 2025 2030 Asp Arg Glu Trp
Cys Phe Thr Gly Glu Arg Asn Asn Gln Ile Leu Glu 2035 2040 2045 Glu
Asn Met Glu Val Glu Ile Trp Thr Arg Glu Gly Glu Lys Lys Lys 2050
2055 2060 Leu Arg Pro Arg Trp Leu Asp Ala Arg Val Tyr Ala Asp Pro
Met Ala2065 2070 2075 2080Leu Lys Asp Phe Lys Glu Phe Ala Ser Gly
Arg Lys Ser Ile Thr Leu 2085 2090 2095 Asp Ile Leu Thr Glu Ile Ala
Ser Leu Pro Thr Tyr Leu Ser Ser Arg 2100 2105 2110 Ala Lys Leu Ala
Leu Asp Asn Ile Val Met Leu His Thr Thr Glu Arg 2115 2120 2125 Gly
Gly Arg Ala Tyr Gln His Ala Leu Asn Glu Leu Pro Glu Ser Leu 2130
2135 2140 Glu Thr Leu Met Leu Val Ala Leu Leu Gly Ala Met Thr Ala
Gly Ile2145 2150 2155 2160Phe Leu Phe Phe Met Gln Gly Lys Gly Ile
Gly Lys Leu Ser Met Gly 2165 2170 2175 Leu Ile Thr Ile Ala Val Ala
Ser Gly Leu Leu Trp Val Ala Glu Ile 2180 2185 2190 Gln Pro Gln Trp
Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Met 2195 2200 2205 Val
Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn 2210
2215 2220 Gln Leu Ile Tyr Val Ile Leu Thr Ile Leu Thr Ile Ile Gly
Leu Ile2225 2230 2235 2240Ala Ala Asn Glu Met Gly Leu Ile Glu Lys
Thr Lys Thr Asp Phe Gly 2245 2250 2255 Phe Tyr Gln Val Lys Thr Glu
Thr Thr Ile Leu Asp Val Asp Leu Arg 2260 2265 2270 Pro Ala Ser Ala
Trp Thr Leu Tyr Ala Val Ala Thr Thr Ile Leu Thr 2275 2280 2285 Pro
Met Leu Arg His Thr Ile Glu Asn Thr Ser Ala Asn Leu Ser Leu 2290
2295 2300 Ala Ala Ile Ala Asn Gln Ala Ala Val Leu Met Gly Leu Gly
Lys Gly2305 2310 2315 2320Trp Pro Leu His Arg Met Asp Leu Gly Val
Pro Leu Leu Ala Met Gly 2325 2330 2335 Cys Tyr Ser Gln Val Asn Pro
Thr Thr Leu Thr Ala Ser Leu Val Met 2340 2345 2350 Leu Leu Val His
Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala 2355 2360 2365 Thr
Arg Glu Ala Gln Lys Arg Thr Ala Ala Gly Ile Met Lys Asn Pro 2370
2375 2380 Thr Val Asp Gly Ile Thr Val Ile Asp Leu Glu Pro Ile Ser
Tyr Asp2385 2390 2395 2400Pro Lys Phe Glu Lys Gln Leu Gly Gln Val
Met Leu Leu Val Leu Cys 2405 2410 2415 Ala Gly Gln Leu Leu Leu Met
Arg Thr Thr Trp Ala Phe Cys Glu Val 2420 2425 2430 Leu Thr Leu Ala
Thr Gly Pro Ile Leu Thr Leu Trp Glu Gly Asn Pro 2435 2440 2445 Gly
Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Thr Ala Asn Ile Phe 2450
2455 2460 Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala Phe Ser Leu
Ile Lys2465 2470 2475 2480Asn Ala Gln Thr Pro Arg Arg Gly Thr Gly
Thr Thr Gly Glu Thr Leu 2485 2490 2495 Gly Glu Lys Trp Lys Arg Gln
Leu Asn Ser Leu Asp Arg Lys Glu Phe 2500 2505 2510 Glu Glu Tyr Lys
Arg Ser Gly Ile Leu Glu Val Asp Arg Thr Glu Ala 2515 2520 2525 Lys
Ser Ala Leu Lys Asp Gly Ser Lys Ile Lys His Ala Val Ser Arg 2530
2535 2540 Gly Ser Ser Lys Ile Arg Trp Ile Val Glu Arg Gly Met Val
Lys Pro2545 2550 2555 2560Lys Gly Lys Val Val Asp Leu Gly Cys Gly
Arg Gly Gly Trp Ser Tyr 2565 2570 2575 Tyr Met Ala Thr Leu Lys Asn
Val Thr Glu Val Lys Gly Tyr Thr Lys 2580 2585 2590 Gly Gly Pro Gly
His Glu Glu Pro Ile Pro Met Ala Thr Tyr Gly Trp 2595 2600 2605 Asn
Leu Val Lys Leu His Ser Gly Val Asp Val Phe Tyr Lys Pro Thr 2610
2615 2620 Glu Gln Val Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser
Ser Asn2625 2630 2635 2640Pro Thr Ile Glu Glu Gly Arg Thr Leu Arg
Val Leu Lys Met Val Glu 2645 2650 2655 Pro Trp Leu Ser Ser Lys Pro
Glu Phe Cys Ile Lys Val Leu Asn Pro 2660 2665 2670 Tyr Met Pro Thr
Val Ile Glu Glu Leu Glu Lys Leu Gln Arg Lys His 2675 2680 2685 Gly
Gly Asn Leu Val Arg Cys Pro Leu Ser Arg Asn Ser Thr His Glu 2690
2695 2700 Met Tyr Trp Val Ser Gly Ala Ser Gly Asn Ile Val Ser Ser
Val Asn2705 2710 2715 2720Thr Thr Ser Lys Met Leu Leu Asn Arg Phe
Thr Thr Arg His Arg Lys 2725 2730 2735 Pro Thr Tyr Glu Lys Asp Val
Asp Leu Gly Ala Gly Thr Arg Ser Val 2740 2745 2750 Ser Thr Glu Thr
Glu Lys Pro Asp Met Thr Ile Ile Gly Arg Arg Leu 2755 2760 2765 Gln
Arg Leu Gln Glu Glu His Lys Glu Thr Trp His Tyr Asp Gln Glu 2770
2775 2780 Asn Pro Tyr Arg Thr Trp Ala Tyr His Gly Ser Tyr Glu Ala
Pro Ser2785 2790 2795 2800Thr Gly Ser Ala Ser Ser Met Val Asn Gly
Val Val Lys Leu Leu Thr 2805 2810 2815 Lys Pro Trp Asp Val Ile Pro
Met Val Thr Gln Leu Ala Met Thr Asp 2820 2825 2830 Thr Thr Pro Phe
Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr 2835 2840 2845 Arg
Thr Pro Gln Pro Lys Pro Gly Thr Arg Met Val Met Thr Thr Thr 2850
2855 2860 Ala Asn Trp Leu Trp Ala Leu Leu Gly Lys Lys Lys Asn Pro
Arg Leu2865 2870 2875 2880Cys Thr Arg Glu Glu Phe Ile Ser Lys Val
Arg Ser Asn Ala Ala Ile 2885 2890 2895 Gly Ala Val Phe Gln Glu Glu
Gln Gly Trp Thr Ser Ala Ser Glu Ala 2900 2905 2910 Val Asn Asp Ser
Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Ala Leu 2915 2920 2925 His
Gln Glu Gly Lys Cys Glu Ser Cys Val Tyr Asn Met Met Gly Lys 2930
2935 2940 Arg Glu Lys Lys Leu Gly Glu Phe Gly Arg Ala Lys Gly Ser
Arg Ala2945 2950 2955 2960Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe
Leu Glu Phe Glu Ala Leu 2965 2970 2975 Gly Phe Leu Asn Glu Asp His
Trp Phe Gly Arg Glu Asn Ser Trp Ser 2980 2985 2990 Gly Val Glu Gly
Glu Gly Leu His Arg Leu Gly Tyr Ile Leu Glu Glu 2995 3000 3005 Ile
Asp Lys Lys Asp Gly Asp Leu Met Tyr Ala Asp Asp Thr Ala Gly 3010
3015 3020 Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln Asn Glu Glu
Leu Ile3025 3030 3035 3040Thr Glu Gln Met Ala Pro His His Lys Ile
Leu Ala Lys Ala Ile Phe 3045 3050 3055 Lys Leu Thr Tyr Gln Asn Lys
Val Val Lys Val Leu Arg Pro Thr Pro 3060 3065 3070 Arg Gly Ala Val
Met Asp Ile Ile Ser Arg Lys Asp Gln Arg Gly Ser 3075 3080 3085 Gly
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Val 3090
3095 3100 Gln Leu Ile Arg Gln Met Glu Ala Glu Gly Val Ile Thr Gln
Asp Asp3105 3110 3115 3120Met Gln Asn Pro Lys Gly Leu Lys Glu Arg
Val Glu Lys Trp Leu Lys 3125 3130 3135 Glu Cys Gly Val Asp Arg Leu
Lys Arg Met Ala Ile Ser Gly Asp Asp 3140 3145 3150 Cys Val Val Lys
Pro Leu Asp Glu Arg Phe Gly Thr Ser Leu Leu Phe 3155 3160 3165 Leu
Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln Trp Glu Pro 3170
3175 3180 Ser Lys Gly Trp Lys Asn Trp Gln Glu Val Pro Phe Cys Ser
His His3185 3190 3195 3200Phe His Lys Ile Phe Met Lys Asp Gly Arg
Ser Leu Val Val Pro Cys 3205 3210 3215 Arg Asn Gln Asp Glu Leu Ile
Gly Arg Ala Arg Ile Ser Gln Gly Ala
3220 3225 3230 Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ala
Tyr Ala Gln 3235 3240 3245 Met Trp Ser Leu Met Tyr Phe His Arg Arg
Asp Leu Arg Leu Ala Ser 3250 3255 3260 Met Ala Ile Cys Ser Ala Val
Pro Thr Glu Trp Phe Pro Thr Ser Arg3265 3270 3275 3280Thr Thr Trp
Ser Ile His Ala His His Gln Trp Met Thr Thr Glu Asp 3285 3290 3295
Met Leu Lys Val Trp Asn Arg Val Trp Ile Glu Asp Asn Pro Asn Met
3300 3305 3310 Thr Asp Lys Thr Pro Val His Ser Trp Glu Asp Ile Pro
Tyr Leu Gly 3315 3320 3325 Lys Arg Glu Asp Leu Trp Cys Gly Ser Leu
Ile Gly Leu Ser Ser Arg 3330 3335 3340 Ala Thr Trp Ala Lys Asn Ile
His Thr Ala Ile Thr Gln Val Arg Asn3345 3350 3355 3360Leu Ile Gly
Lys Glu Glu Tyr Val Asp Tyr Met Pro Val Met Lys Arg 3365 3370 3375
Tyr Ser Ala Pro Ser Glu Ser Glu Gly Val Leu 3380 3385
1410649DNADengue 4 virus strain 2A 14agttgttagt ctgtgtggac
cgacaaggac agttccaaat cggaagcttg cttaacacag 60ttctaacagt ttgtttgaat
agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 120tggttagacc
acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag
180ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta
cggatggtgc 240tagcattcat cacgtttttg cgagtccttt ccatcccacc
aacagcaggg attctgaaga 300gatggggaca gttgaagaaa aataaggcca
tcaagatact gattggattc aggaaggaga 360taggccgcat gctgaacatc
ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct 420tgattcccac
cgtaatggcg ttttccttgt caacaagaga tggcgaaccc ctcatgatag
480tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg
atcaacaaat 540gcactctcat tgccatggac ttgggtgaaa tgtgtgagga
cactgtcacg tataaatgcc 600ccctactggt caataccgaa cctgaagaca
ttgattgctg gtgcaacctc acgtctacct 660gggtcatgta tgggacatgc
acccagagcg gagaacggag acgagagaag cgctcagtag 720ctttaacacc
acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg
780aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac
ccaggattcg 840cgctcttggc aggatttatg gcttatatga ttgggcaaac
aggaatccag cgaactgtct 900tctttgtcct aatgatgctg gtcgccccat
cctacggaat gcgatgcgta ggagtaggaa 960acagagactt tgtggaagga
gtctcaggtg gagcatgggt cgacctggtg ctagaacatg 1020gaggatgcgt
cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga
1080caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca
atatcaaaca 1140taactacggc aacaagatgt ccaacgcaag gagagcctta
tctgaaagag gaacaggacc 1200aacagtacat ttgccggaga gatgtggtag
acagagggtg gggcaatggc tgtggcttgt 1260ttggaaaagg aggagttgtg
acatgtgcga agttttcatg ttcggggaag ataacaggca 1320atttggtcca
aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca
1380cccatgcagt aggaaatgac acatccaatc atggagttac agccatgata
actcccaggt 1440caccatcggt ggaagtcaaa ttgccggact atggagaact
aacactcgat tgtgaaccca 1500ggtctggaat tgactttaat gagatgattc
tgatgaaaat gaaaaagaaa acatggctcg 1560tgcataagca atggtttttg
gatctgcctc ttccatggac agcaggagca gacacatcag 1620aggttcactg
gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac
1680aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc
gctggagcca 1740cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg
acatcttaag tgcaaagtcc 1800gtatggagaa attgagaatc aagggaatgt
catacacgat gtgttcagga aagttttcaa 1860ttgacaaaga gatggcagaa
acacagcatg ggacaacagt ggtgaaagtc aagtatgaag 1920gtgctggagc
tccgtgtaaa gtccccatag agataagaga tgtaaacaag gaaaaagtgg
1980ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgta
accaacatag 2040aattagaacc cccctttggg gacagctaca tagtgatagg
tgttggaaac agcgcattaa 2100cactccattg gttcaggaaa gggagttcca
ttggcaagat gtttgagtcc acatacagag 2160gtgcaaaacg aatggccatt
ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2220tgttcacatc
attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt
2280ttggaggagt ctcatggatg attagaatcc taattgggtt cttagtgttg
tggattggca 2340cgaactcaag gaacacttca atggctatga cgtgcatagc
tgttggagga atcactctgt 2400ttctgggctt cacagttcaa gcagacatgg
gttgtgtggt gtcatggagt gggaaagaat 2460tgaagtgtgg aagcggaatt
tttgtggttg acaacgtgca cacttggaca gaacagtaca 2520aatttcaacc
agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg
2580gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag
caaataacca 2640acgagctaaa ctatgttctc tgggaaggag gacatgacct
cactgtagtg gctggggatg 2700tgaagggggt gttgaccaaa ggcaagagag
cactcacacc cccagtgagt gatctgaaat 2760attcatggaa gacatgggga
aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2820ttttaataga
cggaccagac acctctgaat gccccaatga acgaagagca tggaactctc
2880ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg
aaattccgag 2940aaggaagttc agaagtgtgt gaccacaggt taatgtcagc
tgcaattaaa gatcagaaag 3000ctgtgcatgc tgacatgggt tattggatag
agagctcaaa aaaccagacc tggcagatag 3060agaaagcatc tcttattgaa
gtgaaaacat gtctgtggcc caagacccac acactgtgga 3120gcaatggagt
gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac
3180agcacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac
ttaggcaaat 3240tagagataga ctttggagaa tgccccggaa caacagtcac
aattcaggag gattgtgacc 3300atagaggccc atctttgagg accaccactg
catctggaaa actagtcacg caatggtgct 3360gccgctcctg cacgatgcct
cccttaaggt tcttgggaga agatgggtgc tggtatggga 3420tggagattag
gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg
3480gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg
tttgtggaag 3540aatgcttgag gagaagagtc actaggaaac acatgatatt
agttgtggtg atcactcttt 3600gtgctatcat cctgggaggc ctcacatgga
tggacttact acgagccctc atcatgttgg 3660gggacactat gtctggtaga
ataggaggac agatccacct agccatcatg gcagtgttca 3720agatgtcacc
aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag
3780cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat
gaccttatgg 3840aactcattga tggaatatca ctgggactaa ttttgctaaa
aatagtaaca cagtttgaca 3900acacccaagt gggaacctta gctctttcct
tgactttcat aagatcaaca atgccattgg 3960tcatggcttg gaggaccatt
atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4020ggacaagctg
tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag
4080cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga
agatcttggc 4140ctcttaacga gggcataatg gctgtgggtt tggttagtct
cttaggaagc gctcttttaa 4200agaatgatgt ccctttagct ggcccaatgg
tggcaggagg cttacttctg gcggcttacg 4260tgatgagtgg tagctcagca
gatctgtcac tagagaaggc cgccaacgtg cagtgggatg 4320aaatggcaga
cataacaggc tcaagcccaa tcatagaagt gaagcaggat gaagatggct
4380ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg
aaactggcac 4440tgataacagt gtcaggtctc taccccttgg caattccagt
cacaatgacc ttatggtaca 4500tgtggcaagt gaaaacacaa agatcaggag
ccctgtggga cgtcccctca cccgctgcca 4560ctaaaaaagc cgcactgtct
gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4620aaactcaggt
tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa
4680caagaggatc agtgatctgc cacgagactg ggagattgga gccatcttgg
gctgacgtca 4740ggaatgacat gatatcatac ggtgggggat ggaggcttgg
agacaaatgg gacaaagaag 4800aagacgttca ggtcctcgcc atagaaccag
gaaaaaatcc taaacatgtc caaacgaaac 4860ctggcctttt caagacccta
actggagaaa ttggagcagt aacattagat ttcaaacccg 4920gaacgtctgg
ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg
4980gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa
agaattggag 5040agccagatta tgaagtggat gaggacattt ttcgaaagaa
aagattaact ataatggact 5100tacaccccgg agctggaaag acaaaaagaa
ttcttccatc aatagtgaga gaagccttaa 5160aaaggaggct acgaactttg
attttagctc ccacgagagt ggtggcggcc gagatggaag 5220aggccctacg
tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag
5280gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt
ttgtcatcaa 5340ccagggttcc aaattacaac cttatagtga tggatgaagc
acatttcacc gatccttcta 5400gtgtcgcggc tagaggatac atctcgacca
gggtggaaat gggagaggca gcagccatct 5460tcatgaccgc aacccctccc
ggagcgacag atccctttcc ccagagcaac agcccaatag 5520aagacatcga
gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag
5580actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat
gacattgcaa 5640attgtttgag aaagtcggga aagaaagtta tccagttgag
taggaaaacc tttgatacag 5700agtatccaaa aacgaaactc acggactggg
actttgtggt cactacagac atatctgaaa 5760tgggggccaa ttttagagcc
gggagagtga tagaccctag aagatgcctc aagccagtta 5820tcctaccaga
tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa
5880gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac
gaccaatacg 5940ttttctccgg agacccacta aaaaatgatg aagatcatgc
ccactggaca gaagcaaaga 6000tgctgcttga caatatctac accccagaag
ggatcattcc aacattgttt ggtccggaaa 6060gggaaaaaac ccaagccatt
gatggagagt ttcgcctcag aggggaacaa aggaagactt 6120ttgtggaatt
aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg
6180ctggcatttc ttacaaagat cgggaatggt gcttcacagg ggaaagaaat
aaccaaattt 6240tagaagaaaa catggaggtt gaaatttgga ctagagaggg
agaaaagaaa aagctaaggc 6300caagatggtt agatgcacgt gtatacgctg
accccatggc tttgaaggat ttcaaggagt 6360ttgccagtgg aaggaagagt
ataactctcg acatcctaac agagattgcc agtttgccaa 6420cttacctttc
ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag
6480aaagaggagg gagggcctat caacacgccc tgaacgaact tccggagtca
ctggaaacac 6540tcatgcttgt agctttacta ggtgctatga cagcaggcat
cttcctgttt ttcatgcaag 6600ggaaaggaat agggaaattg tcaatgggtt
tgataaccat tgcggtggct agtggcttgc 6660tctgggtagc agaaattcaa
ccccagtgga tagcggcctc aatcatacta gagttttttc 6720tcatggtact
gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga
6780tctacgtcat attgaccatt ctcaccatca ttggtctaat agcagccaac
gagatggggc 6840tgattgaaaa aacaaaaacg gattttgggt tttaccaggt
aaaaacagaa accaccatcc 6900tcgatgtgga cttgagacca gcttcagcat
ggacgctcta tgcagtagcc accacaattc 6960tgactcccat gctgagacac
accatagaaa acacgtcggc caacctatct ctagcagcca 7020ttgccaacca
ggcagccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg
7080acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca
acaaccttga 7140cagcatcctt agtcatgctt ttagtccatt atgcaataat
aggcccagga ttgcaggcaa 7200aagccacaag agaggcccag aaaaggacag
ctgctgggat catgaaaaat cccacagtgg 7260acgggataac agtaatagat
ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7320tagggcaggt
catgctacta gtcttgtgtg ctggacaact actcttgatg agaacaacat
7380gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg
tgggagggca 7440acccgggaag gttttggaac acgaccatag ccgtatccac
cgccaacatt ttcaggggaa 7500gttacttggc gggagctgga ctggcttttt
cactcataaa gaatgcacaa acccctagga 7560ggggaactgg gaccacagga
gagacactgg gagagaagtg gaagagacag ctaaactcat 7620tagacagaaa
agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg
7680aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatct
agagggtcca 7740gtaagatcag atggattgtt gagagaggga tggtaaagcc
aaaagggaaa gttgtagatc 7800ttggctgtgg gagaggagga tggtcttatt
acatggcgac actcaagaac gtgactgaag 7860tgaaagggta tacaaaagga
ggtccaggac atgaagaacc gattcccatg gctacttatg 7920gttggaattt
ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag
7980tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata
gaggaaggaa 8040gaacattaag agttttgaag atggtggagc catggctctc
ttcaaaacct gaattctgca 8100tcaaagtcct taacccctac atgccaacag
tcatagaaga gctggagaaa ctgcagagaa 8160aacatggtgg gaaccttgtc
agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8220gggtgtcagg
agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt
8280tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta
gatcttgggg 8340caggaacgag aagtgtctcc actgaaacag aaaaaccaga
catgacaatc attgggagaa 8400ggcttcagcg attgcaagaa gagcacaaag
aaacctggca ttatgatcag gaaaacccat 8460acagaacctg ggcgtatcat
ggaagctatg aagctccttc gacaggctct gcatcctcca 8520tggtgaacgg
ggtggtaaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc
8580agttagccat gacagataca accccttttg ggcaacaaag agtgttcaaa
gagaaggtgg 8640ataccagaac accacaacca aaacccggta cacgaatggt
tatgaccacg acagccaatt 8700ggctgtgggc cctccttgga aagaagaaaa
atcccagact gtgcacaagg gaagagttca 8760tctcaaaagt tagatcaaac
gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8820catcagccag
tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg
8880ccctacacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga
aaacgtgaga 8940aaaagttagg agagtttggc agagccaagg gaagccgagc
aatctggtac atgtggctgg 9000gagcgcggtt tctggaattt gaagccctgg
gttttttgaa tgaagatcac tggtttggca 9060gagaaaattc atggagtgga
gtggaagggg aaggtctgca cagattggga tatatcctgg 9120aggagataga
caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca
9180caagaatcac tgaggatgac cttcaaaatg aggaactgat cacggaacag
atggctcccc 9240accacaagat cctagccaaa gccattttca aactaaccta
tcaaaacaaa gtggtgaaag 9300tcctcagacc cacaccgaga ggagcggtga
tggatatcat atccaggaaa gaccaaagag 9360gtagtggaca agttggaaca
tatggtttga acacattcac caacatggaa gttcaactca 9420tccgccaaat
ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt
9480tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta
aagaggatgg 9540caatcagtgg agacgattgc gtggtgaagc ccctagatga
gaggtttggc acttccctcc 9600tcttcttgaa cgacatggga aaggtgagga
aagacattcc gcagtgggaa ccatctaagg 9660gatggaaaaa ctggcaagag
gttccttttt gctcccacca ctttcacaag atctttatga 9720aggatggccg
ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca
9780gaatctcgca gggagctgga tggagcttaa gagaaacagc ctgcctgggc
aaagcttacg 9840cccagatgtg gtcgcttatg tacttccaca gaagggatct
gcgtttagcc tccatggcca 9900tatgctcagc agttccaacg gaatggtttc
caacaagcag aacaacatgg tcaatccacg 9960ctcatcacca gtggatgacc
actgaagata tgctcaaagt gtggaacaga gtgtggatag 10020aagacaaccc
taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc
10080tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc
agagccacct 10140gggcgaagaa cattcacacg gccataaccc aggtcaggaa
cctgatcgga aaagaggaat 10200acgtggatta catgccagta atgaaaagat
acagtgctcc ttcagagagt gaaggagttc 10260tgtaattacc aacaacaaac
accaaaggct attgaagtca ggccacttgt gccacggttt 10320gagcaaaccg
tgctgcctgt agctccgcca ataatgggag gcgtaataat ccccagggag
10380gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga
ggagacccct 10440cccatcactg acaaaacgca gcaaaagggg gcccgaagcc
aggaggaagc tgtactcctg 10500gtggaaggac tagaggttag aggagacccc
cccaacacaa aaacagcata ttgacgctgg 10560gaaagaccag agatcctgct
gtctctgcaa catcaatcca ggcacagagc gccgcaagat 10620ggattggtgt
tgttgatcca acaggttct 10649153387PRTRecombinant Dengue 4 virus
strain rDEN4 15Met Asn Gln Arg Lys Lys Val Val Arg Pro Pro Phe Asn
Met Leu Lys 1 5 10 15 Arg Glu Arg Asn Arg Val Ser Thr Pro Gln Gly
Leu Val Lys Arg Phe 20 25 30 Ser Thr Gly Leu Phe Ser Gly Lys Gly
Pro Leu Arg Met Val Leu Ala 35 40 45 Phe Ile Thr Phe Leu Arg Val
Leu Ser Ile Pro Pro Thr Ala Gly Ile 50 55 60 Leu Lys Arg Trp Gly
Gln Leu Lys Lys Asn Lys Ala Ile Lys Ile Leu65 70 75 80 Ile Gly Phe
Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn Gly 85 90 95 Arg
Lys Arg Ser Thr Ile Thr Leu Leu Cys Leu Ile Pro Thr Val Met 100 105
110 Ala Phe Ser Leu Ser Thr Arg Asp Gly Glu Pro Leu Met Ile Val Ala
115 120 125 Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu
Gly Ile 130 135 140 Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu
Met Cys Glu Asp145 150 155 160 Thr Val Thr Tyr Lys Cys Pro Leu Leu
Val Asn Thr Glu Pro Glu Asp 165 170 175 Ile Asp Cys Trp Cys Asn Leu
Thr Ser Thr Trp Val Met Tyr Gly Thr 180 185 190 Cys Thr Gln Ser Gly
Glu Arg Arg Arg Glu Lys Arg Ser Val Ala Leu 195 200 205 Thr Pro His
Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp Met 210 215 220 Ser
Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp Ile225 230
235 240 Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr
Met 245 250 255 Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val
Leu Met Met 260 265 270 Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val
Gly Val Gly Asn Arg 275 280 285 Asp Phe Val Glu Gly Val Ser Gly Gly
Ala Trp Val Asp Leu Val Leu 290 295 300 Glu His Gly Gly Cys Val Thr
Thr Met Ala Gln Gly Lys Pro Thr Leu305 310 315 320 Asp Phe Glu Leu
Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu Arg 325 330 335 Thr Tyr
Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr Arg 340 345 350
Cys Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln Gln 355
360 365 Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly
Cys 370 375 380 Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys
Phe Ser Cys385 390 395 400 Ser Gly Lys Ile Thr Gly Asn Leu Val Gln
Ile Glu Asn Leu Glu Tyr 405 410 415 Thr Val Val Val Thr Val His Asn
Gly Asp Thr His Ala Val Gly Asn 420 425 430 Asp Thr Ser Asn His Gly
Val Thr Ala Met Ile Thr Pro Arg Ser Pro 435 440 445 Ser Val Glu Val
Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp Cys 450 455 460 Glu Pro
Arg Ser Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys Met465 470 475
480 Lys Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu Pro
485 490 495 Leu Pro Trp Thr
Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn Tyr 500 505 510 Lys Glu
Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gln Asp 515 520 525
Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His Ser Ala Leu Ala 530
535 540 Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala
Gly545 550 555 560 His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg
Ile Lys Gly Met 565 570 575 Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser
Ile Asp Lys Glu Met Ala 580 585 590 Glu Thr Gln His Gly Thr Thr Val
Val Lys Val Lys Tyr Glu Gly Ala 595 600 605 Gly Ala Pro Cys Lys Val
Pro Ile Glu Ile Arg Asp Val Asn Lys Glu 610 615 620 Lys Val Val Gly
Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn Thr625 630 635 640 Asn
Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser Tyr 645 650
655 Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe Arg
660 665 670 Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg
Gly Ala 675 680 685 Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp
Phe Gly Ser Val 690 695 700 Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala
Val His Gln Val Phe Gly705 710 715 720 Ser Val Tyr Thr Thr Met Phe
Gly Gly Val Ser Trp Met Ile Arg Ile 725 730 735 Leu Ile Gly Phe Leu
Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr 740 745 750 Ser Met Ala
Met Thr Cys Ile Ala Val Gly Gly Ile Thr Leu Phe Leu 755 760 765 Gly
Phe Thr Val Gln Ala Asp Met Gly Cys Val Ala Ser Trp Ser Gly 770 775
780 Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Val Asp Asn Val
His785 790 795 800 Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser
Pro Ala Arg Leu 805 810 815 Ala Ser Ala Ile Leu Asn Ala His Lys Asp
Gly Val Cys Gly Ile Arg 820 825 830 Ser Thr Thr Arg Leu Glu Asn Val
Met Trp Lys Gln Ile Thr Asn Glu 835 840 845 Leu Asn Tyr Val Leu Trp
Glu Gly Gly His Asp Leu Thr Val Val Ala 850 855 860 Gly Asp Val Lys
Gly Val Leu Thr Lys Gly Lys Arg Ala Leu Thr Pro865 870 875 880 Pro
Val Ser Asp Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys 885 890
895 Ile Phe Thr Pro Glu Ala Arg Asn Ser Thr Phe Leu Ile Asp Gly Pro
900 905 910 Asp Thr Ser Glu Cys Pro Asn Glu Arg Arg Ala Trp Asn Ser
Leu Glu 915 920 925 Val Glu Asp Tyr Gly Phe Gly Met Phe Thr Thr Asn
Ile Trp Met Lys 930 935 940 Phe Arg Glu Gly Ser Ser Glu Val Cys Asp
His Arg Leu Met Ser Ala945 950 955 960 Ala Ile Lys Asp Gln Lys Ala
Val His Ala Asp Met Gly Tyr Trp Ile 965 970 975 Glu Ser Ser Lys Asn
Gln Thr Trp Gln Ile Glu Lys Ala Ser Leu Ile 980 985 990 Glu Val Lys
Thr Cys Leu Trp Pro Lys Thr His Thr Leu Trp Ser Asn 995 1000 1005
Gly Val Leu Glu Ser Gln Met Leu Ile Pro Lys Ser Tyr Ala Gly Pro
1010 1015 1020 Phe Ser Gln His Asn Tyr Arg Gln Gly Tyr Ala Thr Gln
Thr Val Gly1025 1030 1035 1040Pro Trp His Leu Gly Lys Leu Glu Ile
Asp Phe Gly Glu Cys Pro Gly 1045 1050 1055 Thr Thr Val Thr Ile Gln
Glu Asp Cys Asp His Arg Gly Pro Ser Leu 1060 1065 1070 Arg Thr Thr
Thr Ala Ser Gly Lys Leu Val Thr Gln Trp Cys Cys Arg 1075 1080 1085
Ser Cys Thr Met Pro Pro Leu Arg Phe Leu Gly Glu Asp Gly Cys Trp
1090 1095 1100 Tyr Gly Met Glu Ile Arg Pro Leu Ser Glu Lys Glu Glu
Asn Met Val1105 1110 1115 1120Lys Ser Gln Val Thr Ala Gly Gln Gly
Thr Ser Glu Thr Phe Ser Met 1125 1130 1135 Gly Leu Leu Cys Leu Thr
Leu Phe Val Glu Glu Cys Leu Arg Arg Arg 1140 1145 1150 Val Thr Arg
Lys His Met Ile Leu Val Val Val Ile Thr Leu Cys Ala 1155 1160 1165
Ile Ile Leu Gly Gly Leu Thr Trp Met Asp Leu Leu Arg Ala Leu Ile
1170 1175 1180 Met Leu Gly Asp Thr Met Ser Gly Arg Ile Gly Gly Gln
Ile His Leu1185 1190 1195 1200Ala Ile Met Ala Val Phe Lys Met Ser
Pro Gly Tyr Val Leu Gly Val 1205 1210 1215 Phe Leu Arg Lys Leu Thr
Ser Arg Glu Thr Ala Leu Met Val Ile Gly 1220 1225 1230 Met Ala Met
Thr Thr Val Leu Ser Ile Pro His Asp Leu Met Glu Leu 1235 1240 1245
Ile Asp Gly Ile Ser Leu Gly Leu Ile Leu Leu Lys Ile Val Thr Gln
1250 1255 1260 Phe Asp Asn Thr Gln Val Gly Thr Leu Ala Leu Ser Leu
Thr Phe Ile1265 1270 1275 1280Arg Ser Thr Met Pro Leu Val Met Ala
Trp Arg Thr Ile Met Ala Val 1285 1290 1295 Leu Phe Val Val Thr Leu
Ile Pro Leu Cys Arg Thr Ser Cys Leu Gln 1300 1305 1310 Lys Gln Ser
His Trp Val Glu Ile Thr Ala Leu Ile Leu Gly Ala Gln 1315 1320 1325
Ala Leu Pro Val Tyr Leu Met Thr Leu Met Lys Gly Ala Ser Arg Arg
1330 1335 1340 Ser Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Leu
Val Ser Leu1345 1350 1355 1360Leu Gly Ser Ala Leu Leu Lys Asn Asp
Val Pro Leu Ala Gly Pro Met 1365 1370 1375 Val Ala Gly Gly Leu Leu
Leu Ala Ala Tyr Val Met Ser Gly Ser Ser 1380 1385 1390 Ala Asp Leu
Ser Leu Glu Lys Ala Ala Asn Val Gln Trp Asp Glu Met 1395 1400 1405
Ala Asp Ile Thr Gly Ser Ser Pro Ile Val Glu Val Lys Gln Asp Glu
1410 1415 1420 Asp Gly Ser Phe Ser Ile Arg Asp Val Glu Glu Thr Asn
Met Ile Thr1425 1430 1435 1440Leu Leu Val Lys Leu Ala Leu Ile Thr
Val Ser Gly Leu Tyr Pro Leu 1445 1450 1455 Ala Ile Pro Val Thr Met
Thr Leu Trp Tyr Met Trp Gln Val Lys Thr 1460 1465 1470 Gln Arg Ser
Gly Ala Leu Trp Asp Val Pro Ser Pro Ala Ala Thr Lys 1475 1480 1485
Lys Ala Ala Leu Ser Glu Gly Val Tyr Arg Ile Met Gln Arg Gly Leu
1490 1495 1500 Phe Gly Lys Thr Gln Val Gly Val Gly Ile His Met Glu
Gly Val Phe1505 1510 1515 1520His Thr Met Trp His Val Thr Arg Gly
Ser Val Ile Cys His Glu Thr 1525 1530 1535 Gly Arg Leu Glu Pro Ser
Trp Ala Asp Val Arg Asn Asp Met Ile Ser 1540 1545 1550 Tyr Gly Gly
Gly Trp Arg Leu Gly Asp Lys Trp Asp Lys Glu Glu Asp 1555 1560 1565
Val Gln Val Leu Ala Ile Glu Pro Gly Lys Asn Pro Lys His Val Gln
1570 1575 1580 Thr Lys Pro Gly Leu Phe Lys Thr Leu Thr Gly Glu Ile
Gly Ala Val1585 1590 1595 1600Thr Leu Asp Phe Lys Pro Gly Thr Ser
Gly Ser Pro Ile Ile Asn Arg 1605 1610 1615 Lys Gly Lys Val Ile Gly
Leu Tyr Gly Asn Gly Val Val Thr Lys Ser 1620 1625 1630 Gly Asp Tyr
Val Ser Ala Ile Thr Gln Ala Glu Arg Ile Gly Glu Pro 1635 1640 1645
Asp Tyr Glu Val Asp Glu Asp Ile Phe Arg Lys Lys Arg Leu Thr Ile
1650 1655 1660 Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Ile
Leu Pro Ser1665 1670 1675 1680Ile Val Arg Glu Ala Leu Lys Arg Arg
Leu Arg Thr Leu Ile Leu Ala 1685 1690 1695 Pro 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 Val Lys Ser Glu His Thr Gly Arg 1715 1720 1725
Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Thr Arg Leu Leu
1730 1735 1740 Ser Ser Thr Arg Val Pro Asn Tyr Asn Leu Ile Val Met
Asp Glu Ala1745 1750 1755 1760His Phe Thr Asp Pro Ser Ser Val Ala
Ala Arg Gly Tyr Ile Ser Thr 1765 1770 1775 Arg Val Glu Met Gly Glu
Ala Ala Ala Ile Phe Met Thr Ala Thr Pro 1780 1785 1790 Pro Gly Ala
Thr Asp Pro Phe Pro Gln Ser Asn Ser Pro Ile Glu Asp 1795 1800 1805
Ile Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn Thr Gly Phe Asp Trp
1810 1815 1820 Ile Thr Asp Tyr Gln Gly Lys Thr Val Trp Phe Val Pro
Ser Ile Lys1825 1830 1835 1840Ala Gly Asn Asp Ile Ala Asn Cys Leu
Arg Lys Ser Gly Lys Lys Val 1845 1850 1855 Ile Gln Leu Ser Arg Lys
Thr Phe Asp Thr Glu Tyr Pro Lys Thr Lys 1860 1865 1870 Leu Thr Asp
Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly 1875 1880 1885
Ala Asn Phe Arg Ala Gly Arg Val Ile Asp Pro Arg Arg Cys Leu Lys
1890 1895 1900 Pro Val Ile Leu Pro Asp Gly Pro Glu Arg Val Ile Leu
Ala Gly Pro1905 1910 1915 1920Ile Pro Val Thr Pro Ala Ser Ala Ala
Gln Arg Arg Gly Arg Ile Gly 1925 1930 1935 Arg Asn Pro Ala Gln Glu
Asp Asp Gln Tyr Val Phe Ser Gly Asp Pro 1940 1945 1950 Leu Lys Asn
Asp Glu Asp His Ala His Trp Thr Glu Ala Lys Met Leu 1955 1960 1965
Leu Asp Asn Ile Tyr Thr Pro Glu Gly Ile Ile Pro Thr Leu Phe Gly
1970 1975 1980 Pro Glu Arg Glu Lys Thr Gln Ala Ile Asp Gly Glu Phe
Arg Leu Arg1985 1990 1995 2000Gly Glu Gln Arg Lys Thr Phe Val Glu
Leu Met Arg Arg Gly Asp Leu 2005 2010 2015 Pro Val Trp Leu Ser Tyr
Lys Val Ala Ser Ala Gly Ile Ser Tyr Glu 2020 2025 2030 Asp Arg Glu
Trp Cys Phe Thr Gly Glu Arg Asn Asn Gln Ile Leu Glu 2035 2040 2045
Glu Asn Met Glu Val Glu Ile Trp Thr Arg Glu Gly Glu Lys Lys Lys
2050 2055 2060 Leu Arg Pro Arg Trp Leu Asp Ala Arg Val Tyr Ala Asp
Pro Met Ala2065 2070 2075 2080Leu Lys Asp Phe Lys Glu Phe Ala Ser
Gly Arg Lys Ser Ile Thr Leu 2085 2090 2095 Asp Ile Leu Thr Glu Ile
Ala Ser Leu Pro Thr Tyr Leu Ser Ser Arg 2100 2105 2110 Ala Lys Leu
Ala Leu Asp Asn Ile Val Met Leu His Thr Thr Glu Arg 2115 2120 2125
Gly Gly Arg Ala Tyr Gln His Ala Leu Asn Glu Leu Pro Glu Ser Leu
2130 2135 2140 Glu Thr Leu Met Leu Val Ala Leu Leu Gly Ala Met Thr
Ala Gly Ile2145 2150 2155 2160Phe Leu Phe Phe Met Gln Gly Lys Gly
Ile Gly Lys Leu Ser Met Gly 2165 2170 2175 Leu Ile Thr Ile Ala Val
Ala Ser Gly Leu Leu Trp Val Ala Glu Ile 2180 2185 2190 Gln Pro Gln
Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Met 2195 2200 2205
Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn
2210 2215 2220 Gln Leu Ile Tyr Val Ile Leu Thr Ile Leu Thr Ile Ile
Gly Leu Ile2225 2230 2235 2240Ala Ala Asn Glu Met Gly Leu Ile Glu
Lys Thr Lys Thr Asp Phe Gly 2245 2250 2255 Phe Tyr Gln Val Lys Thr
Glu Thr Thr Ile Leu Asp Val Asp Leu Arg 2260 2265 2270 Pro Ala Ser
Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr Ile Leu Thr 2275 2280 2285
Pro Met Leu Arg His Thr Ile Glu Asn Thr Ser Ala Asn Leu Ser Leu
2290 2295 2300 Ala Ala Ile Ala Asn Gln Ala Ala Val Leu Met Gly Leu
Gly Lys Gly2305 2310 2315 2320Trp Pro Leu His Arg Met Asp Leu Gly
Val Pro Leu Leu Ala Met Gly 2325 2330 2335 Cys Tyr Ser Gln Val Asn
Pro Thr Thr Leu Thr Ala Ser Leu Val Met 2340 2345 2350 Leu Leu Val
His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala 2355 2360 2365
Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala Gly Ile Met Lys Asn Pro
2370 2375 2380 Thr Val Asp Gly Ile Thr Val Ile Asp Leu Glu Pro Ile
Ser Tyr Asp2385 2390 2395 2400Pro Lys Phe Glu Lys Gln Leu Gly Gln
Val Met Leu Leu Val Leu Cys 2405 2410 2415 Ala Gly Gln Leu Leu Leu
Met Arg Thr Thr Trp Ala Phe Cys Glu Val 2420 2425 2430 Leu Thr Leu
Ala Thr Gly Pro Ile Leu Thr Leu Trp Glu Gly Asn Pro 2435 2440 2445
Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Thr Ala Asn Ile Phe
2450 2455 2460 Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala Phe Ser
Leu Ile Lys2465 2470 2475 2480Asn Ala Gln Thr Pro Arg Arg Gly Thr
Gly Thr Thr Gly Glu Thr Leu 2485 2490 2495 Gly Glu Lys Trp Lys Arg
Gln Leu Asn Ser Leu Asp Arg Lys Glu Phe 2500 2505 2510 Glu Glu Tyr
Lys Arg Ser Gly Ile Leu Glu Val Asp Arg Thr Glu Ala 2515 2520 2525
Lys Ser Ala Leu Lys Asp Gly Ser Lys Ile Lys His Ala Val Ser Arg
2530 2535 2540 Gly Ser Ser Lys Ile Arg Trp Ile Val Glu Arg Gly Met
Val Lys Pro2545 2550 2555 2560Lys Gly Lys Val Val Asp Leu Gly Cys
Gly Arg Gly Gly Trp Ser Tyr 2565 2570 2575 Tyr Met Ala Thr Leu Lys
Asn Val Thr Glu Val Lys Gly Tyr Thr Lys 2580 2585 2590 Gly Gly Pro
Gly His Glu Glu Pro Ile Pro Met Ala Thr Tyr Gly Trp 2595 2600 2605
Asn Leu Val Lys Leu His Ser Gly Val Asp Val Phe Tyr Lys Pro Thr
2610 2615 2620 Glu Gln Val Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser
Ser Ser Asn2625 2630 2635 2640Pro Thr Ile Glu Glu Gly Arg Thr Leu
Arg Val Leu Lys Met Val Glu 2645 2650 2655 Pro Trp Leu Ser Ser Lys
Pro Glu Phe Cys Ile Lys Val Leu Asn Pro 2660 2665 2670 Tyr Met Pro
Thr Val Ile Glu Glu Leu Glu Lys Leu Gln Arg Lys His 2675 2680 2685
Gly Gly Asn Leu Val Arg Cys Pro Leu Ser Arg Asn Ser Thr His Glu
2690 2695 2700 Met Tyr Trp Val Ser Gly Ala Ser Gly Asn Ile Val Ser
Ser Val Asn2705 2710 2715 2720Thr Thr Ser Lys Met Leu Leu Asn Arg
Phe Thr Thr Arg His Arg Lys 2725 2730 2735 Pro Thr Tyr Glu Lys Asp
Val Asp Leu Gly Ala Gly Thr Arg Ser Val 2740 2745 2750 Ser Thr Glu
Thr Glu Lys Pro Asp Met Thr Ile Ile Gly Arg Arg Leu 2755 2760 2765
Gln Arg Leu Gln Glu Glu His Lys Glu Thr Trp His Tyr Asp Gln Glu
2770 2775 2780 Asn Pro Tyr Arg Thr Trp Ala Tyr His Gly Ser Tyr Glu
Ala Pro Ser2785 2790 2795 2800Thr Gly Ser Ala Ser Ser Met Val Asn
Gly Val Val Lys Leu Leu Thr 2805 2810
2815 Lys Pro Trp Asp Val Ile Pro Met Val Thr Gln Leu Ala Met Thr
Asp 2820 2825 2830 Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu
Lys Val Asp Thr 2835 2840 2845 Arg Thr Pro Gln Pro Lys Pro Gly Thr
Arg Met Val Met Thr Thr Thr 2850 2855 2860 Ala Asn Trp Leu Trp Ala
Leu Leu Gly Lys Lys Lys Asn Pro Arg Leu2865 2870 2875 2880Cys Thr
Arg Glu Glu Phe Ile Ser Lys Val Arg Ser Asn Ala Ala Ile 2885 2890
2895 Gly Ala Val Phe Gln Glu Glu Gln Gly Trp Thr Ser Ala Ser Glu
Ala 2900 2905 2910 Val Asn Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
Glu Arg Ala Leu 2915 2920 2925 His Gln Glu Gly Lys Cys Glu Ser Cys
Val Tyr Asn Met Met Gly Lys 2930 2935 2940 Arg Glu Lys Lys Leu Gly
Glu Phe Gly Arg Ala Lys Gly Ser Arg Ala2945 2950 2955 2960Ile Trp
Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu 2965 2970
2975 Gly Phe Leu Asn Glu Asp His Trp Phe Gly Arg Glu Asn Ser Trp
Ser 2980 2985 2990 Gly Val Glu Gly Glu Gly Leu His Arg Leu Gly Tyr
Ile Leu Glu Glu 2995 3000 3005 Ile Asp Lys Lys Asp Gly Asp Leu Met
Tyr Ala Asp Asp Thr Ala Gly 3010 3015 3020 Trp Asp Thr Arg Ile Thr
Glu Asp Asp Leu Gln Asn Glu Glu Leu Ile3025 3030 3035 3040Thr Glu
Gln Met Ala Pro His His Lys Ile Leu Ala Lys Ala Ile Phe 3045 3050
3055 Lys Leu Thr Tyr Gln Asn Lys Val Val Lys Val Leu Arg Pro Thr
Pro 3060 3065 3070 Arg Gly Ala Val Met Asp Ile Ile Ser Arg Lys Asp
Gln Arg Gly Ser 3075 3080 3085 Gly Gln Val Gly Thr Tyr Gly Leu Asn
Thr Phe Thr Asn Met Glu Val 3090 3095 3100 Gln Leu Ile Arg Gln Met
Glu Ala Glu Gly Val Ile Thr Gln Asp Asp3105 3110 3115 3120Met Gln
Asn Pro Lys Gly Leu Lys Glu Arg Val Glu Lys Trp Leu Lys 3125 3130
3135 Glu Cys Gly Val Asp Arg Leu Lys Arg Met Ala Ile Ser Gly Asp
Asp 3140 3145 3150 Cys Val Val Lys Pro Leu Asp Glu Arg Phe Gly Thr
Ser Leu Leu Phe 3155 3160 3165 Leu Asn Asp Met Gly Lys Val Arg Lys
Asp Ile Pro Gln Trp Glu Pro 3170 3175 3180 Ser Lys Gly Trp Lys Asn
Trp Gln Glu Val Pro Phe Cys Ser His His3185 3190 3195 3200Phe His
Lys Ile Phe Met Lys Asp Gly Arg Ser Leu Val Val Pro Cys 3205 3210
3215 Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln Gly
Ala 3220 3225 3230 Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys
Ala Tyr Ala Gln 3235 3240 3245 Met Trp Ser Leu Met Tyr Phe His Arg
Arg Asp Leu Arg Leu Ala Ser 3250 3255 3260 Met Ala Ile Cys Ser Ala
Val Pro Thr Glu Trp Phe Pro Thr Ser Arg3265 3270 3275 3280Thr Thr
Trp Ser Ile His Ala His His Gln Trp Met Thr Thr Glu Asp 3285 3290
3295 Met Leu Lys Val Trp Asn Arg Val Trp Ile Glu Asp Asn Pro Asn
Met 3300 3305 3310 Thr Asp Lys Thr Pro Val His Ser Trp Glu Asp Ile
Pro Tyr Leu Gly 3315 3320 3325 Lys Arg Glu Asp Leu Trp Cys Gly Ser
Leu Ile Gly Leu Ser Ser Arg 3330 3335 3340 Ala Thr Trp Ala Lys Asn
Ile His Thr Ala Ile Thr Gln Val Arg Asn3345 3350 3355 3360Leu Ile
Gly Lys Glu Glu Tyr Val Asp Tyr Met Pro Val Met Lys Arg 3365 3370
3375 Tyr Ser Ala Pro Ser Glu Ser Glu Gly Val Leu 3380 3385
1610649DNARecombinant Dengue 4 virus strain rDEN4 16agttgttagt
ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 60ttctaacagt
ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg
120tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca
acccctcaag 180ggttggtgaa gagattctca accggacttt tttctgggaa
aggaccctta cggatggtgc 240tagcattcat cacgtttttg cgagtccttt
ccatcccacc aacagcaggg attctgaaga 300gatggggaca gttgaagaaa
aataaggcca tcaagatact gattggattc aggaaggaga 360taggccgcat
gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct
420tgattcccac cgtaatggcg ttttccctca gcacaagaga tggcgaaccc
ctcatgatag 480tggcaaaaca tgaaaggggg agacctctct tgtttaagac
aacagagggg atcaacaaat 540gcactctcat tgccatggac ttgggtgaaa
tgtgtgagga cactgtcacg tataaatgcc 600ccctactggt caataccgaa
cctgaagaca ttgattgctg gtgcaacctc acgtctacct 660gggtcatgta
tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag
720ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatgg
atgtcatcgg 780aaggggcttg gaagcatgct cagagagtag agagctggat
actcagaaac ccaggattcg 840cgctcttggc aggatttatg gcttatatga
ttgggcaaac aggaatccag cgaactgtct 900tctttgtcct aatgatgctg
gtcgccccat cctacggaat gcgatgcgta ggagtaggaa 960acagagactt
tgtggaagga gtctcaggtg gagcatgggt cgacctggtg ctagaacatg
1020gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa
ctgactaaga 1080caacagccaa ggaagtggct ctgttaagaa cctattgcat
tgaagcctca atatcaaaca 1140taactacggc aacaagatgt ccaacgcaag
gagagcctta tctgaaagag gaacaggacc 1200aacagtacat ttgccggaga
gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1260ttggaaaagg
aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca
1320atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac
aatggagaca 1380cccatgcagt aggaaatgac acatccaatc atggagttac
agccatgata actcccaggt 1440caccatcggt ggaagtcaaa ttgccggact
atggagaact aacactcgat tgtgaaccca 1500ggtctggaat tgactttaat
gagatgattc tgatgaaaat gaaaaagaaa acatggctcg 1560tgcataagca
atggtttttg gatctgcctc ttccatggac agcaggagca gacacatcag
1620aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat
gccaagagac 1680aggatgtgac agtgctggga tctcaggaag gagccatgca
ttctgccctc gctggagcca 1740cagaagtgga ctccggtgat ggaaatcaca
tgtttgcagg acatcttaag tgcaaagtcc 1800gtatggagaa attgagaatc
aagggaatgt catacacgat gtgttcagga aagttttcaa 1860ttgacaaaga
gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag
1920gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtaaacaag
gaaaaagtgg 1980ttgggcgtat catctcatcc acccctttgg ctgagaatac
caacagtgta accaacatag 2040aattagaacc cccctttggg gacagctaca
tagtgatagg tgttggaaac agcgcattaa 2100cactccattg gttcaggaaa
gggagttcca ttggcaagat gtttgagtcc acatacagag 2160gtgcaaaacg
aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac
2220tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat
acaaccatgt 2280ttggaggagt ctcatggatg attagaatcc taattgggtt
cttagtgttg tggattggca 2340cgaactcgag gaacacttca atggctatga
cgtgcatagc tgttggagga atcactctgt 2400ttctgggctt cacagttcaa
gcagacatgg gttgtgtggc gtcatggagt gggaaagaat 2460tgaagtgtgg
aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca
2520aatttcaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc
cacaaagatg 2580gggtctgtgg aattagatca accacgaggc tggaaaatgt
catgtggaag caaataacca 2640acgagctaaa ctatgttctc tgggaaggag
gacatgacct cactgtagtg gctggggatg 2700tgaagggggt gttgaccaaa
ggcaagagag cactcacacc cccagtgagt gatctgaaat 2760attcatggaa
gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat
2820ttttaataga cggaccagac acctctgaat gccccaatga acgaagagca
tggaactctc 2880ttgaggtgga agactatgga tttggcatgt tcacgaccaa
catatggatg aaattccgag 2940aaggaagttc agaagtgtgt gaccacaggt
taatgtcagc tgcaattaaa gatcagaaag 3000ctgtgcatgc tgacatgggt
tattggatag agagctcaaa aaaccagacc tggcagatag 3060agaaagcatc
tcttattgaa gtgaaaacat gtctgtggcc caagacccac acactgtgga
3120gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc
cctttttcac 3180agcacaatta ccgccagggc tatgccacgc aaaccgtggg
cccatggcac ttaggcaaat 3240tagagataga ctttggagaa tgccccggaa
caacagtcac aattcaggag gattgtgacc 3300atagaggccc atctttgagg
accaccactg catctggaaa actagtcacg caatggtgct 3360gccgctcctg
cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga
3420tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag
gtgacggccg 3480gacagggcac atcagaaact ttttctatgg gtctgttgtg
cctgaccttg tttgtggaag 3540aatgcttgag gagaagagtc actaggaaac
acatgatatt agttgtggtg atcactcttt 3600gtgctatcat cctgggaggc
ctcacatgga tggacttact acgagccctc atcatgttgg 3660gggacactat
gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca
3720agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca
agagagacag 3780cactaatggt aataggaatg gccatgacaa cggtgctttc
aattccacat gaccttatgg 3840aactcattga tggaatatca ctgggactaa
ttttgctaaa aatagtaaca cagtttgaca 3900acacccaagt gggaacctta
gctctttcct tgactttcat aagatcaaca atgccattgg 3960tcatggcttg
gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca
4020ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc
atcctaggag 4080cccaagctct gccagtgtac ctaatgactc ttatgaaagg
agcctcaaga agatcttggc 4140ctcttaacga gggcataatg gctgtgggtt
tggttagtct cttaggaagc gctcttttaa 4200agaatgatgt ccctttagct
ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4260tgatgagtgg
tagctcagca gatctgtcac tagagaaggc cgccaacgtg cagtgggatg
4320aaatggcaga cataacaggc tcaagcccaa tcgtagaagt gaagcaggat
gaagatggct 4380ctttctccat acgggacgtc gaggaaacca atatgataac
ccttttggtg aaactggcac 4440tgataacagt gtcaggtctc taccccttgg
caattccagt cacaatgacc ttatggtaca 4500tgtggcaagt gaaaacacaa
agatcaggag ccctgtggga cgtcccctca cccgctgcca 4560ctaaaaaagc
cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga
4620aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg
tggcatgtaa 4680caagaggatc agtgatctgc cacgagactg ggagattgga
gccatcttgg gctgacgtca 4740ggaatgacat gatatcatac ggtgggggat
ggaggcttgg agacaaatgg gacaaagaag 4800aagacgttca ggtcctcgcc
atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4860ctggcctttt
caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg
4920gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc
tatggaaatg 4980gagtagttac caaatcaggt gattacgtca gtgccataac
gcaagccgaa agaattggag 5040agccagatta tgaagtggat gaggacattt
ttcgaaagaa aagattaact ataatggact 5100tacaccccgg agctggaaag
acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5160aaaggaggct
acgaactttg attttagctc ccacgagagt ggtggcggcc gagatggaag
5220aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca
gaacacacag 5280gaagagagat tgtagacctc atgtgtcatg caaccttcac
aacaagactt ttgtcatcaa 5340ccagggttcc aaattacaac cttatagtga
tggatgaagc acatttcacc gatccttcta 5400gtgtcgcggc tagaggatac
atctcgacca gggtggaaat gggagaggca gcagccatct 5460tcatgaccgc
aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag
5520aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac
tggataacag 5580actaccaagg gaaaactgtg tggtttgttc ccagcataaa
agctggaaat gacattgcaa 5640attgtttgag aaagtcggga aagaaagtta
tccagttgag taggaaaacc tttgatacag 5700agtatccaaa aacgaaactc
acggactggg actttgtggt cactacagac atatctgaaa 5760tgggggccaa
ttttagagcc gggagagtga tagaccctag aagatgcctc aagccagtta
5820tcctaccaga tgggccagag agagtcattt tagcaggtcc tattccagtg
actccagcaa 5880gcgctgctca gagaagaggg cgaataggaa ggaacccagc
acaagaagac gaccaatacg 5940ttttctccgg agacccacta aaaaatgatg
aagatcatgc ccactggaca gaagcaaaga 6000tgctgcttga caatatctac
accccagaag ggatcattcc aacattgttt ggtccggaaa 6060gggaaaaaac
ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt
6120ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag
gtagcttctg 6180ctggcatttc ttacgaagat cgggaatggt gcttcacagg
ggaaagaaat aaccaaattt 6240tagaagaaaa catggaggtt gaaatttgga
ctagagaggg agaaaagaaa aagctaaggc 6300caagatggtt agatgcacgt
gtatacgctg accccatggc tttgaaggat ttcaaggagt 6360ttgccagtgg
aaggaagagt ataactctcg acatcctaac agagattgcc agtttgccaa
6420cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc
cacacaacag 6480aaagaggagg gagggcctat caacacgccc tgaacgaact
tccggagtca ctggaaacac 6540tcatgcttgt agctttacta ggtgctatga
cagcaggcat cttcctgttt ttcatgcaag 6600ggaaaggaat agggaaattg
tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6660tctgggtagc
agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc
6720tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac
aatcaattga 6780tctacgtcat attgaccatt ctcaccatca ttggtctaat
agcagccaac gagatggggc 6840tgattgaaaa aacaaaaacg gattttgggt
tttaccaggt aaaaacagaa accaccatcc 6900tcgatgtgga cttgagacca
gcttcagcat ggacgctcta tgcagtagcc accacaattc 6960tgactcccat
gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca
7020ttgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccgctc
cacagaatgg 7080acctcggtgt gccgctgtta gcaatgggat gctattctca
agtgaaccca acaaccttga 7140cagcatcctt agtcatgctt ttagtccatt
atgcaataat aggcccagga ttgcaggcaa 7200aagccacaag agaggcccag
aaaaggacag ctgctgggat catgaaaaat cccacagtgg 7260acgggataac
agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat
7320tagggcaggt catgctacta gtcttgtgtg ctggacaact actcttgatg
agaacaacat 7380gggctttctg tgaagtcttg actttggcca caggaccaat
cttgaccttg tgggagggca 7440acccgggaag gttttggaac acgaccatag
ccgtatccac cgccaacatt ttcaggggaa 7500gttacttggc gggagctgga
ctggcttttt cactcataaa gaatgcacaa acccctagga 7560ggggaactgg
gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat
7620tagacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg
gacaggactg 7680aagccaagtc tgccctgaaa gatgggtcta aaatcaagca
tgcagtatca agagggtcca 7740gtaagatcag atggattgtt gagagaggga
tggtaaagcc aaaagggaaa gttgtagatc 7800ttggctgtgg gagaggagga
tggtcttatt acatggcgac actcaagaac gtgactgaag 7860tgaaagggta
tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg
7920gttggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc
acagagcaag 7980tggacaccct gctctgtgat attggggagt catcttctaa
tccaacaata gaggaaggaa 8040gaacattaag agttttgaag atggtggagc
catggctctc ttcaaaacct gaattctgca 8100tcaaagtcct taacccctac
atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8160aacatggtgg
gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt
8220gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca
aagatgttgt 8280tgaacaggtt cacaacaagg cataggaaac ccacttatga
gaaggacgta gatcttgggg 8340caggaacgag aagtgtctcc actgaaacag
aaaaaccaga catgacaatc attgggagaa 8400ggcttcagcg attgcaagaa
gagcacaaag aaacctggca ttatgatcag gaaaacccat 8460acagaacctg
ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca
8520tggtgaacgg ggtggtaaaa ctgctaacaa aaccctggga tgtgattcca
atggtgactc 8580agttagccat gacagataca accccttttg ggcaacaaag
agtgttcaaa gagaaggtgg 8640ataccagaac accacaacca aaacccggta
cacgaatggt tatgaccacg acagccaatt 8700ggctgtgggc cctccttgga
aagaagaaaa atcccagact gtgcacaagg gaagagttca 8760tctcaaaagt
tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga
8820catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac
aaagaaaggg 8880ccctacacca ggaagggaaa tgtgaatcgt gtgtctataa
catgatggga aaacgtgaga 8940aaaagttagg agagtttggc agagccaagg
gaagccgagc aatctggtac atgtggctgg 9000gagcgcggtt tctggaattt
gaagccctgg gttttttgaa tgaagatcac tggtttggca 9060gagaaaattc
atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg
9120aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca
ggctgggaca 9180caagaatcac tgaggatgac cttcaaaatg aggaactgat
cacggaacag atggctcccc 9240accacaagat cctagccaaa gccattttca
aactaaccta tcaaaacaaa gtggtgaaag 9300tcctcagacc cacaccgcgg
ggagcggtga tggatatcat atccaggaaa gaccaaagag 9360gtagtggaca
agttggaaca tatggtttga acacattcac caacatggaa gttcaactca
9420tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac
ccaaaagggt 9480tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt
cgacaggtta aagaggatgg 9540caatcagtgg agacgattgc gtggtgaagc
ccctagatga gaggtttggc acttccctcc 9600tcttcttgaa cgacatggga
aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9660gatggaaaaa
ctggcaagag gttccttttt gctcccacca ctttcacaag atctttatga
9720aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata
gggagagcca 9780gaatctcgca gggagctgga tggagcttaa gagaaacagc
ctgcctgggc aaagcttacg 9840cccagatgtg gtcgcttatg tacttccaca
gaagggatct gcgtttagcc tccatggcca 9900tatgctcagc agttccaacg
gaatggtttc caacaagcag aacaacatgg tcaatccacg 9960ctcatcacca
gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag
10020aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat
ataccttacc 10080tagggaaaag agaggatttg tggtgtggat ccctgattgg
actttcttcc agagccacct 10140gggcgaagaa cattcatacg gccataaccc
aggtcaggaa cctgatcgga aaagaggaat 10200acgtggatta catgccagta
atgaaaagat acagtgctcc ttcagagagt gaaggagttc 10260tgtaattacc
aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggttt
10320gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat
ccccagggag 10380gccatgcgcc acggaagctg tacgcgtggc atattggact
agcggttaga ggagacccct 10440cccatcactg ataaaacgca gcaaaagggg
gcccgaagcc aggaggaagc tgtactcctg 10500gtggaaggac tagaggttag
aggagacccc cccaacacaa aaacagcata ttgacgctgg 10560gaaagaccag
agatcctgct gtctctgcaa catcaatcca ggcacagagc gccgcaagat
10620ggattggtgt tgttgatcca acaggttct 10649173388PRTRecombinant
Dengue rDEN2/4d30 17Met 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 Val65 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 Ala Gly Met Ile Ile Met Leu Ile 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 Glu Asp Gly 130 135 140 Val Asn Met Cys Thr Leu Met Ala
Met Asp Leu Gly Glu Leu Cys Glu145 150 155 160 Asp Thr Ile Thr Tyr
Lys Cys Pro Leu Leu Arg 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 Trp225 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
Ala Leu Ile Phe Ile Leu Leu 260 265 270 Thr Ala Val 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 Thr305 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 Glu
Ser 340 345 350 Arg Cys Pro Thr Gln Gly Glu Pro Ser 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 Thr385 390 395 400 Cys Lys Lys Asn Met Glu
Gly Lys Val Val Gln Pro Glu Asn Leu Glu 405 410 415 Tyr Thr Ile 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 Ile 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
Gln465 470 475 480 Met Glu Asn 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 Thr545 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 Val 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 Glu625 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 Asn 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
Phe705 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 Asn 740 745 750 Thr Ser Met Ala Met Thr Cys Ile
Ala Val Gly Gly Ile Thr Leu Phe 755 760 765 Leu Gly Phe Thr Val Gln
Ala Asp Met Gly Cys Val Ala Ser Trp Ser 770 775 780 Gly Lys Glu Leu
Lys Cys Gly Ser Gly Ile Phe Val Val Asp Asn Val785 790 795 800 His
Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ala Arg 805 810
815 Leu Ala Ser Ala Ile Leu Asn Ala His Lys Asp Gly Val Cys Gly Ile
820 825 830 Arg Ser Thr Thr Arg Leu Glu Asn Val Met Trp Lys Gln Ile
Thr Asn 835 840 845 Glu Leu Asn Tyr Val Leu Trp Glu Gly Gly His Asp
Leu Thr Val Val 850 855 860 Ala Gly Asp Val Lys Gly Val Leu Thr Lys
Gly Lys Arg Ala Leu Thr865 870 875 880 Pro Pro Val Ser Asp Leu Lys
Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Ile Phe Thr Pro
Glu Ala Arg Asn Ser Thr Phe Leu Ile Asp Gly 900 905 910 Pro Asp Thr
Ser Glu Cys Pro Asn Glu Arg Arg Ala Trp Asn Ser Leu 915 920 925 Glu
Val Glu Asp Tyr Gly Phe Gly Met Phe Thr Thr Asn Ile Trp Met 930 935
940 Lys Phe Arg Glu Gly Ser Ser Glu Val Cys Asp His Arg Leu Met
Ser945 950 955 960 Ala Ala Ile Lys Asp Gln Lys Ala Val His Ala Asp
Met Gly Tyr Trp 965 970 975 Ile Glu Ser Ser Lys Asn Gln Thr Trp Gln
Ile Glu Lys Ala Ser Leu 980 985 990 Ile Glu Val Lys Thr Cys Leu Trp
Pro Lys Thr His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu
Ser Gln Met Leu Ile Pro Lys Ser Tyr Ala Gly 1010 1015 1020 Pro Phe
Ser Gln His Asn Tyr Arg Gln Gly Tyr Ala Thr Gln Thr Val1025 1030
1035 1040Gly Pro Trp His Leu Gly Lys Leu Glu Ile Asp Phe Gly Glu
Cys Pro 1045 1050 1055 Gly Thr Thr Val Thr Ile Gln Glu Asp Cys Asp
His Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Ala Ser Gly
Lys Leu Val Thr Gln Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Met
Pro Pro Leu Arg Phe Leu Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr
Gly Met Glu Ile Arg Pro Leu Ser Glu Lys Glu Glu Asn Met1105 1110
1115 1120Val Lys Ser Gln Val Thr Ala Gly Gln Gly Thr Ser Glu Thr
Phe Ser 1125 1130 1135 Met Gly Leu Leu Cys Leu Thr Leu Phe Val Glu
Glu Cys Leu Arg Arg 1140 1145 1150 Arg Val Thr Arg Lys His Met Ile
Leu Val Val Val Ile Thr Leu Cys 1155 1160 1165 Ala Ile Ile Leu Gly
Gly Leu Thr Trp Met Asp Leu Leu Arg Ala Leu 1170 1175 1180 Ile Met
Leu Gly Asp Thr Met Ser Gly Arg Ile Gly Gly Gln Ile His1185 1190
1195 1200Leu Ala Ile Met Ala Val Phe Lys Met Ser Pro Gly Tyr Val
Leu Gly 1205 1210 1215 Val Phe Leu Arg Lys Leu Thr Ser Arg Glu Thr
Ala Leu Met Val Ile 1220 1225 1230 Gly Met Ala Met Thr Thr Val Leu
Ser Ile Pro His Asp Leu Met Glu 1235 1240 1245 Leu Ile Asp Gly Ile
Ser Leu Gly Leu Ile Leu Leu Lys Ile Val Thr 1250 1255 1260 Gln Phe
Asp Asn Thr Gln Val Gly Thr Leu Ala Leu Ser Leu Thr Phe1265 1270
1275 1280Ile Arg Ser Thr Met Pro Leu Val Met Ala Trp Arg Thr Ile
Met Ala 1285 1290 1295 Val Leu Phe Val Val Thr Leu Ile Pro Leu Cys
Arg Thr Ser Cys Leu 1300 1305 1310 Gln Lys Gln Ser His Trp Val Glu
Ile Thr Ala Leu Ile Leu Gly Ala 1315 1320 1325 Gln Ala Leu Pro Val
Tyr Leu Met Thr Leu Met Lys Gly Ala Ser Arg 1330 1335 1340 Arg Ser
Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Leu Val Ser1345 1350
1355 1360Leu Leu Gly Ser Ala Leu Leu Lys Asn Asp Val Pro Leu Ala
Gly Pro 1365 1370 1375 Met Val Ala Gly Gly Leu Leu Leu Ala Ala Tyr
Val Met Ser Gly Ser 1380 1385 1390 Ser Ala Asp Leu Ser Leu Glu Lys
Ala Ala Asn Val Gln Trp Asp Glu 1395 1400 1405 Met Ala Asp Ile Thr
Gly Ser Ser Pro Ile Val Glu Val Lys Gln Asp 1410 1415 1420 Glu Asp
Gly Ser Phe Ser Ile Arg Asp Val Glu Glu Thr Asn Met Ile1425 1430
1435 1440Thr Leu Leu Val Lys Leu Ala Leu Ile Thr Val Ser Gly Leu
Tyr Pro 1445 1450 1455 Leu Ala Ile Pro Val Thr Met Thr Leu Trp Tyr
Met Trp Gln Val Lys 1460 1465 1470 Thr Gln Arg Ser Gly Ala Leu Trp
Asp Val Pro Ser Pro Ala Ala Thr 1475 1480 1485 Lys Lys Ala Ala Leu
Ser Glu Gly Val Tyr Arg Ile Met Gln Arg Gly 1490 1495 1500 Leu Phe
Gly Lys Thr Gln Val Gly Val Gly Ile His Met Glu Gly Val1505 1510
1515 1520Phe His Thr Met Trp His Val Thr Arg Gly Ser Val Ile Cys
His Glu 1525 1530 1535 Thr Gly Arg Leu Glu Pro Ser Trp Ala Asp Val
Arg Asn Asp Met Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Arg Leu
Gly Asp Lys Trp Asp Lys Glu Glu 1555 1560 1565 Asp Val Gln Val Leu
Ala Ile Glu Pro Gly Lys Asn Pro Lys His Val 1570 1575 1580 Gln Thr
Lys Pro Gly Leu Phe Lys Thr Leu Thr Gly Glu Ile Gly Ala1585 1590
1595 1600Val Thr Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile
Ile Asn 1605 1610 1615 Arg Lys Gly Lys Val Ile Gly Leu Tyr Gly Asn
Gly Val Val Thr Lys 1620 1625 1630 Ser Gly Asp Tyr Val Ser Ala Ile
Thr Gln Ala Glu Arg Ile Gly Glu 1635 1640 1645 Pro Asp Tyr Glu Val
Asp Glu Asp Ile Phe Arg Lys Lys Arg Leu Thr 1650 1655 1660 Ile Met
Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Ile Leu Pro1665 1670
1675 1680Ser Ile Val Arg Glu Ala Leu Lys Arg Arg Leu Arg Thr Leu
Ile Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ala Glu Met Glu
Glu Ala Leu Arg Gly 1700 1705 1710 Leu Pro Ile Arg Tyr Gln Thr Pro
Ala Val Lys Ser Glu His Thr Gly 1715 1720 1725 Arg Glu Ile Val Asp
Leu Met Cys His Ala Thr Phe Thr Thr Arg Leu 1730 1735 1740 Leu Ser
Ser Thr Arg Val Pro Asn Tyr Asn Leu Ile Val Met Asp Glu1745 1750
1755 1760Ala His Phe Thr Asp Pro Ser Ser Val Ala Ala Arg Gly Tyr
Ile Ser 1765 1770 1775 Thr Arg Val Glu Met Gly Glu Ala Ala Ala Ile
Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ala Thr Asp Pro Phe
Pro Gln Ser Asn Ser Pro Ile Glu 1795 1800 1805 Asp Ile Glu Arg Glu
Ile Pro Glu Arg Ser Trp Asn Thr Gly Phe Asp 1810 1815 1820 Trp Ile
Thr Asp Tyr Gln Gly Lys Thr Val Trp Phe Val Pro Ser Ile1825 1830
1835 1840Lys Ala Gly Asn Asp Ile Ala Asn Cys Leu Arg Lys Ser Gly
Lys Lys 1845 1850 1855 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp Thr
Glu Tyr Pro Lys Thr 1860 1865 1870 Lys Leu Thr Asp Trp Asp Phe Val
Val Thr Thr Asp Ile Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe Arg
Ala Gly Arg Val Ile Asp Pro Arg Arg Cys Leu 1890 1895 1900 Lys Pro
Val Ile Leu Pro Asp Gly Pro Glu Arg Val Ile Leu Ala Gly1905 1910
1915 1920Pro Ile Pro Val Thr Pro Ala Ser Ala Ala Gln Arg Arg Gly
Arg Ile 1925 1930 1935 Gly Arg Asn Pro Ala Gln Glu Asp Asp Gln Tyr
Val Phe Ser Gly Asp 1940 1945 1950 Pro Leu Lys Asn Asp Glu Asp His
Ala His Trp Thr Glu Ala Lys Met 1955 1960 1965 Leu Leu Asp Asn Ile
Tyr Thr Pro Glu Gly Ile Ile Pro Thr Leu Phe 1970 1975 1980 Gly Pro
Glu Arg Glu Lys Thr Gln Ala Ile Asp Gly Glu Phe Arg Leu1985 1990
1995 2000Arg Gly Glu Gln Arg Lys Thr Phe Val Glu Leu Met Arg Arg
Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ser Tyr Lys Val Ala Ser
Ala Gly Ile Ser Tyr 2020 2025 2030 Glu Asp Arg Glu Trp Cys Phe Thr
Gly Glu Arg Asn Asn Gln Ile Leu 2035 2040 2045 Glu Glu Asn Met Glu
Val Glu Ile Trp Thr Arg Glu Gly Glu Lys Lys 2050 2055 2060 Lys Leu
Arg Pro Arg Trp Leu Asp Ala Arg Val Tyr Ala Asp Pro Met2065 2070
2075 2080Ala Leu Lys Asp Phe Lys Glu Phe Ala Ser Gly Arg Lys Ser
Ile Thr 2085 2090 2095 Leu Asp Ile Leu Thr Glu Ile Ala Ser Leu Pro
Thr Tyr Leu Ser Ser 2100 2105 2110 Arg Ala Lys Leu Ala Leu Asp Asn
Ile Val Met Leu His Thr Thr Glu 2115 2120 2125 Arg Gly Gly Arg Ala
Tyr Gln His Ala Leu Asn Glu Leu Pro Glu Ser 2130 2135 2140 Leu Glu
Thr Leu Met Leu Val Ala Leu Leu Gly Ala Met Thr Ala Gly2145 2150
2155 2160Ile Phe Leu Phe Phe Met Gln Gly Lys Gly Ile Gly Lys Leu
Ser Met 2165 2170 2175 Gly Leu Ile Thr Ile Ala Val Ala Ser Gly Leu
Leu Trp Val Ala Glu 2180 2185 2190 Ile Gln Pro Gln Trp Ile Ala Ala
Ser Ile Ile Leu Glu Phe Phe Leu 2195 2200 2205 Met Val Leu Leu Ile
Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp 2210 2215 2220 Asn Gln
Leu Ile Tyr Val Ile Leu Thr Ile Leu Thr Ile Ile Gly Leu2225 2230
2235 2240Ile Ala Ala Asn Glu Met Gly Leu Ile Glu Lys Thr Lys Thr
Asp Phe 2245 2250 2255 Gly Phe Tyr Gln Val Lys Thr Glu Thr Thr Ile
Leu Asp Val Asp Leu 2260 2265 2270 Arg Pro Ala Ser Ala Trp Thr Leu
Tyr Ala Val Ala Thr Thr Ile Leu 2275 2280 2285 Thr Pro Met Leu Arg
His Thr Ile Glu Asn Thr Ser Ala Asn Leu Ser 2290 2295 2300 Leu Ala
Ala Ile Ala Asn Gln Ala Ala Val Leu Met Gly Leu Gly Lys2305 2310
2315 2320Gly Trp Pro Leu His Arg Met Asp Leu Gly Val Pro Leu Leu
Ala Met 2325 2330 2335 Gly Cys Tyr Ser Gln Val Asn Pro Thr Thr Leu
Thr Ala Ser Leu Val 2340 2345 2350 Met Leu Leu Val His Tyr Ala Ile
Ile Gly Pro Gly Leu Gln Ala Lys 2355 2360 2365 Ala Thr Arg Glu Ala
Gln Lys Arg Thr Ala Ala Gly Ile Met Lys Asn 2370 2375 2380 Pro Thr
Val Asp Gly Ile Thr Val Ile Asp Leu Glu Pro Ile Ser Tyr2385 2390
2395 2400Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val
Met Leu Leu Val Leu 2405 2410 2415 Cys Ala Gly Gln Leu Leu Leu Met
Arg Thr Thr Trp Ala Phe Cys Glu 2420 2425 2430 Val Leu Thr Leu Ala
Thr Gly Pro Ile Leu Thr Leu Trp Glu Gly Asn 2435 2440 2445 Pro Gly
Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Thr Ala Asn Ile 2450 2455
2460 Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala Phe Ser Leu
Ile2465 2470 2475 2480Lys Asn Ala Gln Thr Pro Arg Arg Gly Thr Gly
Thr Thr Gly Glu Thr 2485 2490 2495 Leu Gly Glu Lys Trp Lys Arg Gln
Leu Asn Ser Leu Asp Arg Lys Glu 2500 2505 2510 Phe Glu Glu Tyr Lys
Arg Ser Gly Ile Leu Glu Val Asp Arg Thr Glu 2515 2520 2525 Ala Lys
Ser Ala Leu Lys Asp Gly Ser Lys Ile Lys His Ala Val Ser 2530 2535
2540 Arg Gly Ser Ser Lys Ile Arg Trp Ile Val Glu Arg Gly Met Val
Lys2545 2550 2555 2560Pro Lys Gly Lys Val Val Asp Leu Gly Cys Gly
Arg Gly Gly Trp Ser 2565 2570 2575 Tyr Tyr Met Ala Thr Leu Lys Asn
Val Thr Glu Val Lys Gly Tyr Thr 2580 2585 2590 Lys Gly Gly Pro Gly
His Glu Glu Pro Ile Pro Met Ala Thr Tyr Gly 2595 2600 2605 Trp Asn
Leu Val Lys Leu His Ser Gly Val Asp Val Phe Tyr Lys Pro 2610 2615
2620 Thr Glu Gln Val Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser
Ser2625 2630 2635 2640Asn Pro Thr Ile Glu Glu Gly Arg Thr Leu Arg
Val Leu Lys Met Val 2645 2650 2655 Glu Pro Trp Leu Ser Ser Lys Pro
Glu Phe Cys Ile Lys Val Leu Asn 2660 2665 2670 Pro Tyr Met Pro Thr
Val Ile Glu Glu Leu Glu Lys Leu Gln Arg Lys 2675 2680 2685 His Gly
Gly Asn Leu Val Arg Cys Pro Leu Ser Arg Asn Ser Thr His 2690 2695
2700 Glu Met Tyr Trp Val Ser Gly Ala Ser Gly Asn Ile Val Ser Ser
Val2705 2710 2715 2720Asn Thr Thr Ser Lys Met Leu Leu Asn Arg Phe
Thr Thr Arg His Arg 2725 2730 2735 Lys Pro Thr Tyr Glu Lys Asp Val
Asp Leu Gly Ala Gly Thr Arg Ser 2740 2745 2750 Val Ser Thr Glu Thr
Glu Lys Pro Asp Met Thr Ile Ile Gly Arg Arg 2755 2760 2765 Leu Gln
Arg Leu Gln Glu Glu His Lys Glu Thr Trp His Tyr Asp Gln 2770 2775
2780 Glu Asn Pro Tyr Arg Thr Trp Ala Tyr His Gly Ser Tyr Glu Ala
Pro2785 2790 2795 2800Ser Thr Gly Ser Ala Ser Ser Met Val Asn Gly
Val Val Lys Leu Leu 2805 2810 2815 Thr Lys Pro Trp Asp Val Ile Pro
Met Val Thr Gln Leu Ala Met Thr 2820 2825 2830 Asp Thr Thr Pro Phe
Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp 2835 2840 2845 Thr Arg
Thr Pro Gln Pro Lys Pro Gly Thr Arg Met Val Met Thr Thr 2850 2855
2860 Thr Ala Asn Trp Leu Trp Ala Leu Leu Gly Lys Lys Lys Asn Pro
Arg2865 2870 2875 2880Leu Cys Thr Arg Glu Glu Phe Ile Ser Lys Val
Arg Ser Asn Ala Ala 2885 2890 2895 Ile Gly Ala Val Phe Gln Glu Glu
Gln Gly Trp Thr Ser Ala Ser Glu 2900 2905 2910 Ala Val Asn Asp Ser
Arg Phe Trp Glu Leu Val Asp Lys Glu Arg Ala 2915 2920 2925 Leu His
Gln Glu Gly Lys Cys Glu Ser Cys Val Tyr Asn Met Met Gly 2930 2935
2940 Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Arg Ala Lys Gly Ser
Arg2945 2950 2955 2960Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe
Leu Glu Phe Glu Ala 2965 2970 2975 Leu Gly Phe Leu Asn Glu Asp His
Trp Phe Gly Arg Glu Asn Ser Trp 2980 2985 2990 Ser Gly Val Glu Gly
Glu Gly Leu His Arg Leu Gly Tyr Ile Leu Glu 2995 3000 3005 Glu Ile
Asp Lys Lys Asp Gly Asp Leu Met Tyr Ala Asp Asp Thr Ala 3010 3015
3020 Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln Asn Glu Glu
Leu3025 3030 3035 3040Ile Thr Glu Gln Met Ala Pro His His Lys Ile
Leu Ala Lys Ala Ile 3045 3050 3055 Phe Lys Leu Thr Tyr Gln Asn Lys
Val Val Lys Val Leu Arg Pro Thr 3060 3065 3070 Pro Arg Gly Ala Val
Met Asp Ile Ile Ser Arg Lys Asp Gln Arg Gly 3075 3080 3085 Ser Gly
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu 3090 3095
3100 Val Gln Leu Ile Arg Gln Met Glu Ala Glu Gly Val Ile Thr Gln
Asp3105 3110 3115 3120Asp Met Gln Asn Pro Lys Gly Leu Lys Glu Arg
Val Glu Lys Trp Leu 3125 3130 3135 Lys Glu Cys Gly Val Asp Arg Leu
Lys Arg Met Ala Ile Ser Gly Asp 3140 3145 3150 Asp Cys Val Val Lys
Pro Leu Asp Glu Arg Phe Gly Thr Ser Leu Leu 3155 3160 3165 Phe Leu
Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln Trp Glu 3170 3175
3180 Pro Ser Lys Gly Trp Lys Asn Trp Gln Glu Val Pro Phe Cys Ser
His3185 3190 3195 3200His Phe His Lys Ile Phe Met Lys Asp Gly Arg
Ser Leu Val Val Pro 3205 3210 3215 Cys Arg Asn Gln Asp Glu Leu Ile
Gly Arg Ala Arg Ile Ser Gln Gly 3220 3225 3230 Ala Gly Trp Ser Leu
Arg Glu Thr Ala Cys Leu Gly Lys Ala Tyr Ala 3235 3240 3245 Gln Met
Trp Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala 3250 3255
3260 Ser Met Ala Ile Cys Ser Ala Val Pro Thr Glu Trp Phe Pro Thr
Ser3265 3270 3275 3280Arg Thr Thr Trp Ser Ile His Ala His His Gln
Trp Met Thr Thr Glu 3285 3290 3295 Asp Met Leu Lys Val Trp Asn Arg
Val Trp Ile Glu Asp Asn Pro Asn 3300 3305 3310 Met Thr Asp Lys Thr
Pro Val His Ser Trp Glu Asp Ile Pro Tyr Leu 3315 3320 3325 Gly Lys
Arg Glu Asp Leu Trp Cys Gly Ser Leu Ile Gly Leu Ser Ser 3330 3335
3340 Arg Ala Thr Trp Ala Lys Asn Ile His Thr Ala Ile Thr Gln Val
Arg3345 3350 3355 3360Asn Leu Ile Gly Lys Glu Glu Tyr Val Asp Tyr
Met Pro Val Met Lys 3365 3370 3375 Arg Tyr Ser Ala Pro Ser Glu Ser
Glu Gly Val Leu 3380 3385 1810616DNARecombinant dengue virus
rDEN2/4d30 18agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg
cttaacacag 60ttctaacagt ttgtttgaat agagagcaga tctctgatga ataaccaacg
aaaaaaggcg 120agaaatacgc ctttcaatat gctgaaacgc gagagaaacc
gcgtgtcgac tgtacaacag 180ctgacaaaga gattctcact tggaatgctg
cagggacgag gaccattaaa actgttcatg 240gccctggtgg cgttccttcg
tttcctaaca atcccaccaa cagcagggat actgaagaga 300tggggaacaa
ttaaaaaatc aaaagccatt aatgttttga gagggttcag gaaagagatt
360ggaaggatgc tgaacatctt gaacaggaga cgcagaactg caggcatgat
cattatgctg 420attccaacag tgatggcgtt ccatttaacc acacgtaacg
gagaaccaca catgatcgtc 480agtagacaag agaaagggaa aagtcttctg
tttaaaacag aggatggtgt gaacatgtgt 540accctcatgg ccatggacct
tggtgaattg tgtgaagata caatcacgta caagtgtcct 600cttctcaggc
agaatgaacc agaagacata gattgttggt gcaactctac gtccacatgg
660gtaacttatg ggacgtgtac caccacagga gaacacagaa gagaaaaaag
atcagtggca 720ctcgttccac atgtgggaat gggactggag acacgaactg
aaacatggat gtcatcagaa 780ggggcctgga aacatgccca gagaattgaa
acttggatct tgagacatcc aggctttacc 840ataatggcag caatcctggc
atacaccata ggaacgacac atttccaaag agccctgatt 900ttcatcttac
tgacagctgt cgctccttca atgacaatgc gttgcatagg aatatcaaat
960agagactttg tagaaggggt ttcaggagga agctgggttg acatagtctt
agaacatgga 1020agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg
attttgaact gataaaaaca 1080gaagccaaac aacctgccac tctaaggaag
tactgtatag aggcaaagct gaccaacaca 1140acaacagaat ctcgctgccc
aacacaagga gaacctagcc taaatgaaga gcaggacaaa 1200aggttcgtct
gcaaacactc catggtggac agaggatggg gaaatggatg tggattattt
1260ggaaaaggag gcattgtgac ctgtgctatg ttcacatgca aaaagaacat
ggaaggaaaa 1320gtcgtgcaac cagaaaactt ggaatacacc attgtgataa
cacctcactc aggggaagag 1380catgcagtcg gaaatgacac aggaaaacat
ggcaaggaaa tcaaaataac accacagagt 1440tccatcacag aagcagagtt
gacaggctat ggcactgtca cgatggagtg ctctccgaga 1500acgggcctcg
acttcaatga gatggtgttg ctgcaaatgg aaaataaagc ttggctggtg
1560cacaggcaat ggttcctaga cctgccgttg ccatggctgc ccggagcgga
cacacaagga 1620tcaaattgga tacagaaaga gacattggtc actttcaaaa
atccccatgc gaagaaacag 1680gatgttgttg ttttgggatc ccaagaaggg
gccatgcaca cagcactcac aggggccaca 1740gaaatccaga tgtcatcagg
aaacttactg ttcacaggac atctcaagtg caggctgagg 1800atggacaaac
tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt
1860gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtaca
atatgaaggg 1920gacggttctc catgtaagat cccttttgag ataatggatt
tggaaaaaag acatgtttta 1980ggtcgcctga ttacagtcaa cccaatcgta
acagaaaaag atagcccagt caacatagaa 2040gcagaacctc cattcggaga
cagctacatc atcataggag tagagccggg acaattgaag 2100ctcaactggt
ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgagggga
2160gcgaagagaa tggccatttt aggtgacaca gcttgggatt ttggatccct
gggaggagtg 2220tttacatcta taggaaaggc tctccaccaa gttttcggag
caatctatgg ggctgccttc 2280agtggggtct catggactat gaaaatcctc
ataggagtca ttatcacatg gataggaatg 2340aactcgagga acacttcaat
ggctatgacg tgcatagctg ttggaggaat cactctgttt 2400ctgggcttca
cagttcaagc agacatgggt tgtgtggcgt catggagtgg gaaagaattg
2460aagtgtggaa gcggaatttt tgtggttgac aacgtgcaca cttggacaga
acagtacaaa 2520tttcaaccag agtccccagc gagactagcg tctgcaatat
taaatgccca caaagatggg 2580gtctgtggaa ttagatcaac cacgaggctg
gaaaatgtca tgtggaagca aataaccaac 2640gagctaaact atgttctctg
ggaaggagga catgacctca ctgtagtggc tggggatgtg 2700aagggggtgt
tgaccaaagg caagagagca ctcacacccc cagtgagtga tctgaaatat
2760tcatggaaga catggggaaa agcaaaaatc ttcaccccag aagcaagaaa
tagcacattt 2820ttaatagacg gaccagacac ctctgaatgc cccaatgaac
gaagagcatg gaactctctt 2880gaggtggaag actatggatt tggcatgttc
acgaccaaca tatggatgaa attccgagaa 2940ggaagttcag aagtgtgtga
ccacaggtta atgtcagctg caattaaaga tcagaaagct 3000gtgcatgctg
acatgggtta ttggatagag agctcaaaaa accagacctg gcagatagag
3060aaagcatctc ttattgaagt gaaaacatgt ctgtggccca agacccacac
actgtggagc 3120aatggagtgc tggaaagcca gatgctcatt ccaaaatcat
atgcgggccc tttttcacag 3180cacaattacc gccagggcta tgccacgcaa
accgtgggcc catggcactt aggcaaatta 3240gagatagact ttggagaatg
ccccggaaca acagtcacaa ttcaggagga ttgtgaccat 3300agaggcccat
ctttgaggac caccactgca tctggaaaac tagtcacgca atggtgctgc
3360cgctcctgca cgatgcctcc cttaaggttc ttgggagaag atgggtgctg
gtatgggatg 3420gagattaggc ccttgagtga aaaagaagag aacatggtca
aatcacaggt gacggccgga 3480cagggcacat cagaaacttt ttctatgggt
ctgttgtgcc tgaccttgtt tgtggaagaa 3540tgcttgagga gaagagtcac
taggaaacac atgatattag ttgtggtgat cactctttgt 3600gctatcatcc
tgggaggcct cacatggatg gacttactac gagccctcat catgttgggg
3660gacactatgt ctggtagaat aggaggacag atccacctag ccatcatggc
agtgttcaag 3720atgtcaccag gatacgtgct gggtgtgttt ttaaggaaac
tcacttcaag agagacagca 3780ctaatggtaa taggaatggc catgacaacg
gtgctttcaa ttccacatga ccttatggaa 3840ctcattgatg gaatatcact
gggactaatt ttgctaaaaa tagtaacaca gtttgacaac 3900acccaagtgg
gaaccttagc tctttccttg actttcataa gatcaacaat gccattggtc
3960atggcttgga ggaccattat ggctgtgttg tttgtggtca cactcattcc
tttgtgcagg 4020acaagctgtc ttcaaaaaca gtctcattgg gtagaaataa
cagcactcat cctaggagcc 4080caagctctgc cagtgtacct aatgactctt
atgaaaggag cctcaagaag atcttggcct 4140cttaacgagg gcataatggc
tgtgggtttg gttagtctct taggaagcgc tcttttaaag 4200aatgatgtcc
ctttagctgg cccaatggtg gcaggaggct tacttctggc ggcttacgtg
4260atgagtggta gctcagcaga tctgtcacta gagaaggccg ccaacgtgca
gtgggatgaa 4320atggcagaca taacaggctc aagcccaatc atagaagtga
agcaggatga agatggctct 4380ttctccatac gggacgtcga ggaaaccaat
atgataaccc ttttggtgaa actggcactg 4440ataacagtgt caggtctcta
ccccttggca attccagtca caatgacctt atggtacatg 4500tggcaagtga
aaacacaaag atcaggagcc ctgtgggacg tcccctcacc cgctgccact
4560aaaaaagccg cactgtctga aggagtgtac aggatcatgc aaagagggtt
attcgggaaa 4620actcaggttg gagtagggat acacatggaa ggtgtatttc
acacaatgtg gcatgtaaca 4680agaggatcag tgatctgcca cgagactggg
agattggagc catcttgggc tgacgtcagg 4740aatgacatga tatcatacgg
tgggggatgg aggcttggag acaaatggga caaagaagaa 4800gacgttcagg
tcctcgccat agaaccagga aaaaatccta aacatgtcca aacgaaacct
4860ggccttttca agaccctaac tggagaaatt ggagcagtaa cattagattt
caaacccgga 4920acgtctggtt ctcccatcat caacaggaaa ggaaaagtca
tcggactcta tggaaatgga 4980gtagttacca aatcaggtga ttacgtcagt
gccataacgc aagccgaaag aattggagag 5040ccagattatg aagtggatga
ggacattttt cgaaagaaaa gattaactat aatggactta 5100caccccggag
ctggaaagac aaaaagaatt cttccatcaa tagtgagaga agccttaaaa
5160aggaggctac gaactttgat tttagctccc acgagagtgg tggcggccga
gatggaagag 5220gccctacgtg gactgccaat ccgttatcag accccagctg
tgaaatcaga acacacagga 5280agagagattg tagacctcat gtgtcatgca
accttcacaa caagactttt gtcatcaacc 5340agggttccaa attacaacct
tatagtgatg gatgaagcac atttcaccga tccttctagt 5400gtcgcggcta
gaggatacat ctcgaccagg gtggaaatgg gagaggcagc agccatcttc
5460atgaccgcaa cccctcccgg agcgacagat ccctttcccc agagcaacag
cccaatagaa 5520gacatcgaga gggaaattcc ggaaaggtca tggaacacag
ggttcgactg gataacagac 5580taccaaggga aaactgtgtg gtttgttccc
agcataaaag ctggaaatga cattgcaaat 5640tgtttgagaa agtcgggaaa
gaaagttatc cagttgagta ggaaaacctt tgatacagag 5700tatccaaaaa
cgaaactcac ggactgggac tttgtggtca ctacagacat atctgaaatg
5760ggggccaatt ttagagccgg gagagtgata gaccctagaa gatgcctcaa
gccagttatc 5820ctaccagatg ggccagagag agtcatttta gcaggtccta
ttccagtgac tccagcaagc 5880gctgctcaga gaagagggcg aataggaagg
aacccagcac aagaagacga ccaatacgtt 5940ttctccggag acccactaaa
aaatgatgaa gatcatgccc actggacaga agcaaagatg 6000ctgcttgaca
atatctacac cccagaaggg atcattccaa cattgtttgg tccggaaagg
6060gaaaaaaccc aagccattga tggagagttt cgcctcagag gggaacaaag
gaagactttt 6120gtggaattaa tgaggagagg agaccttccg gtgtggctga
gctataaggt agcttctgct 6180ggcatttctt acaaagatcg ggaatggtgc
ttcacagggg aaagaaataa ccaaatttta 6240gaagaaaaca tggaggttga
aatttggact agagagggag aaaagaaaaa gctaaggcca 6300agatggttag
atgcacgtgt atacgctgac cccatggctt tgaaggattt caaggagttt
6360gccagtggaa ggaagagtat aactctcgac atcctaacag agattgccag
tttgccaact 6420tacctttcct ctagggccaa gctcgccctt gataacatag
tcatgctcca cacaacagaa 6480agaggaggga gggcctatca acacgccctg
aacgaacttc cggagtcact ggaaacactc 6540atgcttgtag ctttactagg
tgctatgaca gcaggcatct tcctgttttt catgcaaggg 6600aaaggaatag
ggaaattgtc aatgggtttg ataaccattg cggtggctag tggcttgctc
6660tgggtagcag aaattcaacc ccagtggata gcggcctcaa tcatactaga
gttttttctc 6720atggtactgt tgataccgga accagaaaaa caaaggaccc
cacaagacaa tcaattgatc 6780tacgtcatat tgaccattct caccatcatt
ggtctaatag cagccaacga gatggggctg 6840attgaaaaaa caaaaacgga
ttttgggttt taccaggtaa aaacagaaac caccatcctc 6900gatgtggact
tgagaccagc ttcagcatgg acgctctatg cagtagccac cacaattctg
6960actcccatgc tgagacacac catagaaaac acgtcggcca acctatctct
agcagccatt 7020gccaaccagg cagccgtcct aatggggctt ggaaaaggat
ggccgctcca cagaatggac 7080ctcggtgtgc cgctgttagc aatgggatgc
tattctcaag tgaacccaac aaccttgaca 7140gcatccttag tcatgctttt
agtccattat gcaataatag gcccaggatt gcaggcaaaa 7200gccacaagag
aggcccagaa aaggacagct gctgggatca tgaaaaatcc cacagtggac
7260gggataacag taatagatct agaaccaata tcctatgacc caaaatttga
aaagcaatta 7320gggcaggtca tgctactagt cttgtgtgct ggacaactac
tcttgatgag aacaacatgg 7380gctttctgtg aagtcttgac tttggccaca
ggaccaatct tgaccttgtg ggagggcaac 7440ccgggaaggt tttggaacac
gaccatagcc gtatccaccg ccaacatttt caggggaagt 7500tacttggcgg
gagctggact ggctttttca ctcataaaga atgcacaaac ccctaggagg
7560ggaactggga ccacaggaga gacactggga gagaagtgga agagacagct
aaactcatta 7620gacagaaaag agtttgaaga gtataaaaga agtggaatac
tagaagtgga caggactgaa 7680gccaagtctg ccctgaaaga tgggtctaaa
atcaagcatg cagtatcaag agggtccagt 7740aagatcagat ggattgttga
gagagggatg gtaaagccaa aagggaaagt tgtagatctt 7800ggctgtggga
gaggaggatg gtcttattac atggcgacac tcaagaacgt gactgaagtg
7860aaagggtata caaaaggagg tccaggacat gaagaaccga ttcccatggc
tacttatggt 7920tggaatttgg tcaaactcca ttcaggggtt gacgtgttct
acaaacccac agagcaagtg 7980gacaccctgc tctgtgatat tggggagtca
tcttctaatc caacaataga ggaaggaaga 8040acattaagag ttttgaagat
ggtggagcca tggctctctt caaaacctga attctgcatc 8100aaagtcctta
acccctacat gccaacagtc atagaagagc tggagaaact gcagagaaaa
8160catggtggga accttgtcag atgcccgctg tccaggaact ccacccatga
gatgtattgg 8220gtgtcaggag cgtcgggaaa cattgtgagc tctgtgaaca
caacatcaaa gatgttgttg 8280aacaggttca caacaaggca taggaaaccc
acttatgaga aggacgtaga tcttggggca 8340ggaacgagaa gtgtctccac
tgaaacagaa aaaccagaca tgacaatcat tgggagaagg 8400cttcagcgat
tgcaagaaga gcacaaagaa acctggcatt atgatcagga aaacccatac
8460agaacctggg cgtatcatgg aagctatgaa gctccttcga caggctctgc
atcctccatg 8520gtgaacgggg tggtaaaact gctaacaaaa ccctgggatg
tgattccaat ggtgactcag 8580ttagccatga cagatacaac cccttttggg
caacaaagag tgttcaaaga
gaaggtggat 8640accagaacac cacaaccaaa acccggtaca cgaatggtta
tgaccacgac agccaattgg 8700ctgtgggccc tccttggaaa gaagaaaaat
cccagactgt gcacaaggga agagttcatc 8760tcaaaagtta gatcaaacgc
agccataggc gcagtctttc aggaagaaca gggatggaca 8820tcagccagtg
aagctgtgaa tgacagccgg ttttgggaac tggttgacaa agaaagggcc
8880ctacaccagg aagggaaatg tgaatcgtgt gtctataaca tgatgggaaa
acgtgagaaa 8940aagttaggag agtttggcag agccaaggga agccgagcaa
tctggtacat gtggctggga 9000gcgcggtttc tggaatttga agccctgggt
tttttgaatg aagatcactg gtttggcaga 9060gaaaattcat ggagtggagt
ggaaggggaa ggtctgcaca gattgggata tatcctggag 9120gagatagaca
agaaggatgg agacctaatg tatgctgatg acacagcagg ctgggacaca
9180agaatcactg aggatgacct tcaaaatgag gaactgatca cggaacagat
ggctccccac 9240cacaagatcc tagccaaagc cattttcaaa ctaacctatc
aaaacaaagt ggtgaaagtc 9300ctcagaccca caccgcgggg agcggtgatg
gatatcatat ccaggaaaga ccaaagaggt 9360agtggacaag ttggaacata
tggtttgaac acattcacca acatggaagt tcaactcatc 9420cgccaaatgg
aagctgaagg agtcatcaca caagatgaca tgcagaaccc aaaagggttg
9480aaagaaagag ttgagaaatg gctgaaagag tgtggtgtcg acaggttaaa
gaggatggca 9540atcagtggag acgattgcgt ggtgaagccc ctagatgaga
ggtttggcac ttccctcctc 9600ttcttgaacg acatgggaaa ggtgaggaaa
gacattccgc agtgggaacc atctaaggga 9660tggaaaaact ggcaagaggt
tcctttttgc tcccaccact ttcacaagat ctttatgaag 9720gatggccgct
cactagttgt tccatgtaga aaccaggatg aactgatagg gagagccaga
9780atctcgcagg gagctggatg gagcttaaga gaaacagcct gcctgggcaa
agcttacgcc 9840cagatgtggt cgcttatgta cttccacaga agggatctgc
gtttagcctc catggccata 9900tgctcagcag ttccaacgga atggtttcca
acaagcagaa caacatggtc aatccacgct 9960catcaccagt ggatgaccac
tgaagatatg ctcaaagtgt ggaacagagt gtggatagaa 10020gacaacccta
atatgactga caagactcca gtccattcgt gggaagatat accttaccta
10080gggaaaagag aggatttgtg gtgtggatcc ctgattggac tttcttccag
agccacctgg 10140gcgaagaaca ttcacacggc cataacccag gtcaggaacc
tgatcggaaa agaggaatac 10200gtggattaca tgccagtaat gaaaagatac
agtgctcctt cagagagtga aggagttctg 10260taattaccaa caacaaacac
caaaggctat tgaagtcagg ccacttgtgc cacggtttga 10320gcaaaccgtg
ctgcctgtag ctccgccaat aatgggaggc gtaataatcc ccagggaggc
10380catgcgccac ggaagctgta cgcgtggcat attggactag cggttagagg
agacccctcc 10440catcactgac aaaacgcagc aaaagggggc ccaagactag
aggttagagg agaccccccc 10500aacacaaaaa cagcatattg acgctgggaa
agaccagaga tcctgctgtc tctgcaacat 10560caatccaggc acagagcgcc
gcaagatgga ttggtgttgt tgatccaaca ggttct 10616193387PRTDengue 4
virus 19Met Asn Gln Arg Lys Lys Val Val Arg Pro Pro Phe Asn Met Leu
Lys 1 5 10 15 Arg Glu Arg Asn Arg Val Ser Thr Pro Gln Gly Leu Val
Lys Arg Phe 20 25 30 Ser Thr Gly Leu Phe Ser Gly Lys Gly Pro Leu
Arg Met Val Leu Ala 35 40 45 Phe Ile Thr Phe Leu Arg Val Leu Ser
Ile Pro Pro Thr Ala Gly Ile 50 55 60 Leu Lys Arg Trp Gly Gln Leu
Lys Lys Asn Lys Ala Ile Lys Ile Leu65 70 75 80 Ile Gly Phe Arg Lys
Glu Ile Gly Arg Met Leu Asn Ile Leu Asn Gly 85 90 95 Arg Lys Arg
Ser Thr Ile Thr Leu Leu Cys Leu Ile Pro Thr Val Met 100 105 110 Ala
Phe Ser Leu Ser Thr Arg Asp Gly Glu Pro Leu Met Ile Val Ala 115 120
125 Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly Ile
130 135 140 Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys
Glu Asp145 150 155 160 Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn
Thr Glu Pro Glu Asp 165 170 175 Ile Asp Cys Trp Cys Asn Leu Thr Ser
Thr Trp Val Met Tyr Gly Thr 180 185 190 Cys Thr Gln Ser Gly Glu Arg
Arg Arg Glu Lys Arg Ser Val Ala Leu 195 200 205 Thr Pro His Ser Gly
Met Gly Leu Glu Thr Arg Ala Glu Thr Trp Met 210 215 220 Ser Ser Glu
Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp Ile225 230 235 240
Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr Met 245
250 255 Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met
Met 260 265 270 Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val
Gly Asn Arg 275 280 285 Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp
Val Asp Leu Val Leu 290 295 300 Glu His Gly Gly Cys Val Thr Thr Met
Ala Gln Gly Lys Pro Thr Leu305 310 315 320 Asp Phe Glu Leu Thr Lys
Thr Thr Ala Lys Glu Val Ala Leu Leu Arg 325 330 335 Thr Tyr Cys Ile
Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr Arg 340 345 350 Cys Pro
Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln Gln 355 360 365
Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly Cys 370
375 380 Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser
Cys385 390 395 400 Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu
Asn Leu Glu Tyr 405 410 415 Thr Val Val Val Thr Val His Asn Gly Asp
Thr His Ala Val Gly Asn 420 425 430 Asp Thr Ser Asn His Gly Val Thr
Ala Met Ile Thr Pro Arg Ser Pro 435 440 445 Ser Val Glu Val Lys Leu
Pro Asp Tyr Gly Glu Leu Thr Leu Asp Cys 450 455 460 Glu Pro Arg Ser
Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys Met465 470 475 480 Lys
Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu Pro 485 490
495 Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn Tyr
500 505 510 Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg
Gln Asp 515 520 525 Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His
Ser Ala Leu Ala 530 535 540 Gly Ala Thr Glu Val Asp Ser Gly Asp Gly
Asn His Met Phe Ala Gly545 550 555 560 His Leu Lys Cys Lys Val Arg
Met Glu Lys Leu Arg Ile Lys Gly Met 565 570 575 Ser Tyr Thr Met Cys
Ser Gly Lys Phe Ser Ile Asp Lys Glu Met Ala 580 585 590 Glu Thr Gln
His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly Ala 595 600 605 Gly
Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys Glu 610 615
620 Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn
Thr625 630 635 640 Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe
Gly Asp Ser Tyr 645 650 655 Ile Val Ile Gly Val Gly Asn Ser Ala Leu
Thr Leu His Trp Phe Arg 660 665 670 Lys Gly Ser Ser Ile Gly Lys Met
Phe Glu Ser Thr Tyr Arg Gly Ala 675 680 685 Lys Arg Met Ala Ile Leu
Gly Glu Thr Ala Trp Asp Phe Gly Ser Val 690 695 700 Gly Gly Leu Phe
Thr Ser Leu Gly Lys Ala Val His Gln Val Phe Gly705 710 715 720 Ser
Val Tyr Thr Thr Met Phe Gly Gly Val Ser Trp Met Ile Arg Ile 725 730
735 Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn Thr
740 745 750 Ser Met Ala Met Thr Cys Ile Ala Val Gly Gly Ile Thr Leu
Phe Leu 755 760 765 Gly Phe Thr Val Gln Ala Asp Met Gly Cys Val Ala
Ser Trp Ser Gly 770 775 780 Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe
Val Val Asp Asn Val His785 790 795 800 Thr Trp Thr Glu Gln Tyr Lys
Phe Gln Pro Glu Ser Pro Ala Arg Leu 805 810 815 Ala Ser Ala Ile Leu
Asn Ala His Lys Asp Gly Val Cys Gly Ile Arg 820 825 830 Ser Thr Thr
Arg Leu Glu Asn Val Met Trp Lys Gln Ile Thr Asn Glu 835 840 845 Leu
Asn Tyr Val Leu Trp Glu Gly Gly His Asp Leu Thr Val Val Ala 850 855
860 Gly Asp Val Lys Gly Val Leu Thr Lys Gly Lys Arg Ala Leu Thr
Pro865 870 875 880 Pro Val Ser Asp Leu Lys Tyr Ser Trp Lys Thr Trp
Gly Lys Ala Lys 885 890 895 Ile Phe Thr Pro Glu Ala Arg Asn Ser Thr
Phe Leu Ile Asp Gly Pro 900 905 910 Asp Thr Ser Glu Cys Pro Asn Glu
Arg Arg Ala Trp Asn Ser Leu Glu 915 920 925 Val Glu Asp Tyr Gly Phe
Gly Met Phe Thr Thr Asn Ile Trp Met Lys 930 935 940 Phe Arg Glu Gly
Ser Ser Glu Val Cys Asp His Arg Leu Met Ser Ala945 950 955 960 Ala
Ile Lys Asp Gln Lys Ala Val His Ala Asp Met Gly Tyr Trp Ile 965 970
975 Glu Ser Ser Lys Asn Gln Thr Trp Gln Ile Glu Lys Ala Ser Leu Ile
980 985 990 Glu Val Lys Thr Cys Leu Trp Pro Lys Thr His Thr Leu Trp
Ser Asn 995 1000 1005 Gly Val Leu Glu Ser Gln Met Leu Ile Pro Lys
Ser Tyr Ala Gly Pro 1010 1015 1020 Phe Ser Gln His Asn Tyr Arg Gln
Gly Tyr Ala Thr Gln Thr Val Gly1025 1030 1035 1040Pro Trp His Leu
Gly Lys Leu Glu Ile Asp Phe Gly Glu Cys Pro Gly 1045 1050 1055 Thr
Thr Val Thr Ile Gln Glu Asp Cys Asp His Arg Gly Pro Ser Leu 1060
1065 1070 Arg Thr Thr Thr Ala Ser Gly Lys Leu Val Thr Gln Trp Cys
Cys Arg 1075 1080 1085 Ser Cys Thr Met Pro Pro Leu Arg Phe Leu Gly
Glu Asp Gly Cys Trp 1090 1095 1100 Tyr Gly Met Glu Ile Arg Pro Leu
Ser Glu Lys Glu Glu Asn Met Val1105 1110 1115 1120Lys Ser Gln Val
Thr Ala Gly Gln Gly Thr Ser Glu Thr Phe Ser Met 1125 1130 1135 Gly
Leu Leu Cys Leu Thr Leu Phe Val Glu Glu Cys Leu Arg Arg Arg 1140
1145 1150 Val Thr Arg Lys His Met Ile Leu Val Val Val Ile Thr Leu
Cys Ala 1155 1160 1165 Ile Ile Leu Gly Gly Leu Thr Trp Met Asp Leu
Leu Arg Ala Leu Ile 1170 1175 1180 Met Leu Gly Asp Thr Met Ser Gly
Arg Ile Gly Gly Gln Ile His Leu1185 1190 1195 1200Ala Ile Met Ala
Val Phe Lys Met Ser Pro Gly Tyr Val Leu Gly Val 1205 1210 1215 Phe
Leu Arg Lys Leu Thr Ser Arg Glu Thr Ala Leu Met Val Ile Gly 1220
1225 1230 Met Ala Met Thr Thr Val Leu Ser Ile Pro His Asp Leu Met
Glu Leu 1235 1240 1245 Ile Asp Gly Ile Ser Leu Gly Leu Ile Leu Leu
Lys Ile Val Thr Gln 1250 1255 1260 Phe Asp Asn Thr Gln Val Gly Thr
Leu Ala Leu Ser Leu Thr Phe Ile1265 1270 1275 1280Arg Ser Thr Met
Pro Leu Val Met Ala Trp Arg Thr Ile Met Ala Val 1285 1290 1295 Leu
Phe Val Val Thr Leu Ile Pro Leu Cys Arg Thr Ser Cys Leu Gln 1300
1305 1310 Lys Gln Ser His Trp Val Glu Ile Thr Ala Leu Ile Leu Gly
Ala Gln 1315 1320 1325 Ala Leu Pro Val Tyr Leu Met Thr Leu Met Lys
Gly Ala Ser Arg Arg 1330 1335 1340 Ser Trp Pro Leu Asn Glu Gly Ile
Met Ala Val Gly Leu Val Ser Leu1345 1350 1355 1360Leu Gly Ser Ala
Leu Leu Lys Asn Asp Val Pro Leu Ala Gly Pro Met 1365 1370 1375 Val
Ala Gly Gly Leu Leu Leu Ala Ala Tyr Val Met Ser Gly Ser Ser 1380
1385 1390 Ala Asp Leu Ser Leu Glu Lys Ala Ala Asn Val Gln Trp Asp
Glu Met 1395 1400 1405 Ala Asp Ile Thr Gly Ser Ser Pro Ile Ile Glu
Val Lys Gln Asp Glu 1410 1415 1420 Asp Gly Ser Phe Ser Ile Arg Asp
Val Glu Glu Thr Asn Met Ile Thr1425 1430 1435 1440Leu Leu Val Lys
Leu Ala Leu Ile Thr Val Ser Gly Leu Tyr Pro Leu 1445 1450 1455 Ala
Ile Pro Val Thr Met Thr Leu Trp Tyr Met Trp Gln Val Lys Thr 1460
1465 1470 Gln Arg Ser Gly Ala Leu Trp Asp Val Pro Ser Pro Ala Ala
Thr Lys 1475 1480 1485 Lys Ala Ala Leu Ser Glu Gly Val Tyr Arg Ile
Met Gln Arg Gly Leu 1490 1495 1500 Phe Gly Lys Thr Gln Val Gly Val
Gly Ile His Met Glu Gly Val Phe1505 1510 1515 1520His Thr Met Trp
His Val Thr Arg Gly Ser Val Ile Cys His Glu Thr 1525 1530 1535 Gly
Arg Leu Glu Pro Ser Trp Ala Asp Val Arg Asn Asp Met Ile Ser 1540
1545 1550 Tyr Gly Gly Gly Trp Arg Leu Gly Asp Lys Trp Asp Lys Glu
Glu Asp 1555 1560 1565 Val Gln Val Leu Ala Ile Glu Pro Gly Lys Asn
Pro Lys His Val Gln 1570 1575 1580 Thr Lys Pro Gly Leu Phe Lys Thr
Leu Thr Gly Glu Ile Gly Ala Val1585 1590 1595 1600Thr Leu Asp Phe
Lys Pro Gly Thr Ser Gly Ser Pro Ile Ile Asn Arg 1605 1610 1615 Lys
Gly Lys Val Ile Gly Leu Tyr Gly Asn Gly Val Val Thr Lys Ser 1620
1625 1630 Gly Asp Tyr Val Ser Ala Ile Thr Gln Ala Glu Arg Ile Gly
Glu Pro 1635 1640 1645 Asp Tyr Glu Val Asp Glu Asp Ile Phe Arg Lys
Lys Arg Leu Thr Ile 1650 1655 1660 Met Asp Leu His Pro Gly Ala Gly
Lys Thr Lys Arg Ile Leu Pro Ser1665 1670 1675 1680Ile Val Arg Glu
Ala Leu Lys Arg Arg Leu Arg Thr Leu Ile Leu Ala 1685 1690 1695 Pro
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 Val Lys Ser Glu His Thr
Gly Arg 1715 1720 1725 Glu Ile Val Asp Leu Met Cys His Ala Thr Phe
Thr Thr Arg Leu Leu 1730 1735 1740 Ser Ser Thr Arg Val Pro Asn Tyr
Asn Leu Ile Val Met Asp Glu Ala1745 1750 1755 1760His Phe Thr Asp
Pro Ser Ser Val Ala Ala Arg Gly Tyr Ile Ser Thr 1765 1770 1775 Arg
Val Glu Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro 1780
1785 1790 Pro Gly Ala Thr Asp Pro Phe Pro Gln Ser Asn Ser Pro Ile
Glu Asp 1795 1800 1805 Ile Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn
Thr Gly Phe Asp Trp 1810 1815 1820 Ile Thr Asp Tyr Gln Gly Lys Thr
Val Trp Phe Val Pro Ser Ile Lys1825 1830 1835 1840Ala Gly Asn Asp
Ile Ala Asn Cys Leu Arg Lys Ser Gly Lys Lys Val 1845 1850 1855 Ile
Gln Leu Ser Arg Lys Thr Phe Asp Thr Glu Tyr Pro Lys Thr Lys 1860
1865 1870 Leu Thr Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu
Met Gly 1875 1880 1885 Ala Asn Phe Arg Ala Gly Arg Val Ile Asp Pro
Arg Arg Cys Leu Lys 1890 1895 1900 Pro Val Ile Leu Pro Asp Gly Pro
Glu Arg Val Ile Leu Ala Gly Pro1905 1910 1915 1920Ile Pro Val Thr
Pro Ala Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly 1925 1930 1935 Arg
Asn Pro Ala Gln Glu Asp Asp Gln Tyr Val Phe Ser Gly Asp Pro 1940
1945 1950 Leu Lys Asn Asp Glu Asp His Ala His Trp Thr Glu Ala Lys
Met Leu 1955 1960 1965 Leu Asp Asn Ile Tyr Thr Pro Glu Gly Ile Ile
Pro Thr Leu Phe Gly 1970 1975 1980 Pro Glu Arg Glu Lys Thr Gln Ala
Ile Asp Gly Glu Phe Arg Leu Arg1985 1990 1995 2000Gly Glu Gln Arg
Lys Thr Phe Val Glu Leu Met
Arg Arg Gly Asp Leu 2005 2010 2015 Pro Val Trp Leu Ser Tyr Lys Val
Ala Ser Ala Gly Ile Ser Tyr Lys 2020 2025 2030 Asp Arg Glu Trp Cys
Phe Thr Gly Glu Arg Asn Asn Gln Ile Leu Glu 2035 2040 2045 Glu Asn
Met Glu Val Glu Ile Trp Thr Arg Glu Gly Glu Lys Lys Lys 2050 2055
2060 Leu Arg Pro Arg Trp Leu Asp Ala Arg Val Tyr Ala Asp Pro Met
Ala2065 2070 2075 2080Leu Lys Asp Phe Lys Glu Phe Ala Ser Gly Arg
Lys Ser Ile Thr Leu 2085 2090 2095 Asp Ile Leu Thr Glu Ile Ala Ser
Leu Pro Thr Tyr Leu Ser Ser Arg 2100 2105 2110 Ala Lys Leu Ala Leu
Asp Asn Ile Val Met Leu His Thr Thr Glu Arg 2115 2120 2125 Gly Gly
Arg Ala Tyr Gln His Ala Leu Asn Glu Leu Pro Glu Ser Leu 2130 2135
2140 Glu Thr Leu Met Leu Val Ala Leu Leu Gly Ala Met Thr Ala Gly
Ile2145 2150 2155 2160Phe Leu Phe Phe Met Gln Gly Lys Gly Ile Gly
Lys Leu Ser Met Gly 2165 2170 2175 Leu Ile Thr Ile Ala Val Ala Ser
Gly Leu Leu Trp Val Ala Glu Ile 2180 2185 2190 Gln Pro Gln Trp Ile
Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Met 2195 2200 2205 Val Leu
Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn 2210 2215
2220 Gln Leu Ile Tyr Val Ile Leu Thr Ile Leu Thr Ile Ile Gly Leu
Ile2225 2230 2235 2240Ala Ala Asn Glu Met Gly Leu Ile Glu Lys Thr
Lys Thr Asp Phe Gly 2245 2250 2255 Phe Tyr Gln Val Lys Thr Glu Thr
Thr Ile Leu Asp Val Asp Leu Arg 2260 2265 2270 Pro Ala Ser Ala Trp
Thr Leu Tyr Ala Val Ala Thr Thr Ile Leu Thr 2275 2280 2285 Pro Met
Leu Arg His Thr Ile Glu Asn Thr Ser Ala Asn Leu Ser Leu 2290 2295
2300 Ala Ala Ile Ala Asn Gln Ala Ala Val Leu Met Gly Leu Gly Lys
Gly2305 2310 2315 2320Trp Pro Leu His Arg Met Asp Leu Gly Val Pro
Leu Leu Ala Met Gly 2325 2330 2335 Cys Tyr Ser Gln Val Asn Pro Thr
Thr Leu Thr Ala Ser Leu Val Met 2340 2345 2350 Leu Leu Val His Tyr
Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala 2355 2360 2365 Thr Arg
Glu Ala Gln Lys Arg Thr Ala Ala Gly Ile Met Lys Asn Pro 2370 2375
2380 Thr Val Asp Gly Ile Thr Val Ile Asp Leu Glu Pro Ile Ser Tyr
Asp2385 2390 2395 2400Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met
Leu Leu Val Leu Cys 2405 2410 2415 Ala Gly Gln Leu Leu Leu Met Arg
Thr Thr Trp Ala Phe Cys Glu Val 2420 2425 2430 Leu Thr Leu Ala Thr
Gly Pro Ile Leu Thr Leu Trp Glu Gly Asn Pro 2435 2440 2445 Gly Arg
Phe Trp Asn Thr Thr Ile Ala Val Ser Thr Ala Asn Ile Phe 2450 2455
2460 Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Ala Phe Ser Leu Ile
Lys2465 2470 2475 2480Asn Ala Gln Thr Pro Arg Arg Gly Thr Gly Thr
Thr Gly Glu Thr Leu 2485 2490 2495 Gly Glu Lys Trp Lys Arg Gln Leu
Asn Ser Leu Asp Arg Lys Glu Phe 2500 2505 2510 Glu Glu Tyr Lys Arg
Ser Gly Ile Leu Glu Val Asp Arg Thr Glu Ala 2515 2520 2525 Lys Ser
Ala Leu Lys Asp Gly Ser Lys Ile Lys His Ala Val Ser Arg 2530 2535
2540 Gly Ser Ser Lys Ile Arg Trp Ile Val Glu Arg Gly Met Val Lys
Pro2545 2550 2555 2560Lys Gly Lys Val Val Asp Leu Gly Cys Gly Arg
Gly Gly Trp Ser Tyr 2565 2570 2575 Tyr Met Ala Thr Leu Lys Asn Val
Thr Glu Val Lys Gly Tyr Thr Lys 2580 2585 2590 Gly Gly Pro Gly His
Glu Glu Pro Ile Pro Met Ala Thr Tyr Gly Trp 2595 2600 2605 Asn Leu
Val Lys Leu His Ser Gly Val Asp Val Phe Tyr Lys Pro Thr 2610 2615
2620 Glu Gln Val Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Ser
Asn2625 2630 2635 2640Pro Thr Ile Glu Glu Gly Arg Thr Leu Arg Val
Leu Lys Met Val Glu 2645 2650 2655 Pro Trp Leu Ser Ser Lys Pro Glu
Phe Cys Ile Lys Val Leu Asn Pro 2660 2665 2670 Tyr Met Pro Thr Val
Ile Glu Glu Leu Glu Lys Leu Gln Arg Lys His 2675 2680 2685 Gly Gly
Asn Leu Val Arg Cys Pro Leu Ser Arg Asn Ser Thr His Glu 2690 2695
2700 Met Tyr Trp Val Ser Gly Ala Ser Gly Asn Ile Val Ser Ser Val
Asn2705 2710 2715 2720Thr Thr Ser Lys Met Leu Leu Asn Arg Phe Thr
Thr Arg His Arg Lys 2725 2730 2735 Pro Thr Tyr Glu Lys Asp Val Asp
Leu Gly Ala Gly Thr Arg Ser Val 2740 2745 2750 Ser Thr Glu Thr Glu
Lys Pro Asp Met Thr Ile Ile Gly Arg Arg Leu 2755 2760 2765 Gln Arg
Leu Gln Glu Glu His Lys Glu Thr Trp His Tyr Asp Gln Glu 2770 2775
2780 Asn Pro Tyr Arg Thr Trp Ala Tyr His Gly Ser Tyr Glu Ala Pro
Ser2785 2790 2795 2800Thr Gly Ser Ala Ser Ser Met Val Asn Gly Val
Val Lys Leu Leu Thr 2805 2810 2815 Lys Pro Trp Asp Val Ile Pro Met
Val Thr Gln Leu Ala Met Thr Asp 2820 2825 2830 Thr Thr Pro Phe Gly
Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr 2835 2840 2845 Arg Thr
Pro Gln Pro Lys Pro Gly Thr Arg Met Val Met Thr Thr Thr 2850 2855
2860 Ala Asn Trp Leu Trp Ala Leu Leu Gly Lys Lys Lys Asn Pro Arg
Leu2865 2870 2875 2880Cys Thr Arg Glu Glu Phe Ile Ser Lys Val Arg
Ser Asn Ala Ala Ile 2885 2890 2895 Gly Ala Val Phe Gln Glu Glu Gln
Gly Trp Thr Ser Ala Ser Glu Ala 2900 2905 2910 Val Asn Asp Ser Arg
Phe Trp Glu Leu Val Asp Lys Glu Arg Ala Leu 2915 2920 2925 His Gln
Glu Gly Lys Cys Glu Ser Cys Val Tyr Asn Met Met Gly Lys 2930 2935
2940 Arg Glu Lys Lys Leu Gly Glu Phe Gly Arg Ala Lys Gly Ser Arg
Ala2945 2950 2955 2960Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu
Glu Phe Glu Ala Leu 2965 2970 2975 Gly Phe Leu Asn Glu Asp His Trp
Phe Gly Arg Glu Asn Ser Trp Ser 2980 2985 2990 Gly Val Glu Gly Glu
Gly Leu His Arg Leu Gly Tyr Ile Leu Glu Glu 2995 3000 3005 Ile Asp
Lys Lys Asp Gly Asp Leu Met Tyr Ala Asp Asp Thr Ala Gly 3010 3015
3020 Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln Asn Glu Glu Leu
Ile3025 3030 3035 3040Thr Glu Gln Met Ala Pro His His Lys Ile Leu
Ala Lys Ala Ile Phe 3045 3050 3055 Lys Leu Thr Tyr Gln Asn Lys Val
Val Lys Val Leu Arg Pro Thr Pro 3060 3065 3070 Arg Gly Ala Val Met
Asp Ile Ile Ser Arg Lys Asp Gln Arg Gly Ser 3075 3080 3085 Gly Gln
Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Val 3090 3095
3100 Gln Leu Ile Arg Gln Met Glu Ala Glu Gly Val Ile Thr Gln Asp
Asp3105 3110 3115 3120Met Gln Asn Pro Lys Gly Leu Lys Glu Arg Val
Glu Lys Trp Leu Lys 3125 3130 3135 Glu Cys Gly Val Asp Arg Leu Lys
Arg Met Ala Ile Ser Gly Asp Asp 3140 3145 3150 Cys Val Val Lys Pro
Leu Asp Glu Arg Phe Gly Thr Ser Leu Leu Phe 3155 3160 3165 Leu Asn
Asp Met Gly Lys Val Arg Lys Asp Ile Pro Gln Trp Glu Pro 3170 3175
3180 Ser Lys Gly Trp Lys Asn Trp Gln Glu Val Pro Phe Cys Ser His
His3185 3190 3195 3200Phe His Lys Ile Phe Met Lys Asp Gly Arg Ser
Leu Val Val Pro Cys 3205 3210 3215 Arg Asn Gln Asp Glu Leu Ile Gly
Arg Ala Arg Ile Ser Gln Gly Ala 3220 3225 3230 Gly Trp Ser Leu Arg
Glu Thr Ala Cys Leu Gly Lys Ala Tyr Ala Gln 3235 3240 3245 Met Trp
Ser Leu Met Tyr Phe His Arg Arg Asp Leu Arg Leu Ala Ser 3250 3255
3260 Met Ala Ile Cys Ser Ala Val Pro Thr Glu Trp Phe Pro Thr Ser
Arg3265 3270 3275 3280Thr Thr Trp Ser Ile His Ala His His Gln Trp
Met Thr Thr Glu Asp 3285 3290 3295 Met Leu Lys Val Trp Asn Arg Val
Trp Ile Glu Asp Asn Pro Asn Met 3300 3305 3310 Thr Asp Lys Thr Pro
Val His Ser Trp Glu Asp Ile Pro Tyr Leu Gly 3315 3320 3325 Lys Arg
Glu Asp Leu Trp Cys Gly Ser Leu Ile Gly Leu Ser Ser Arg 3330 3335
3340 Ala Thr Trp Ala Lys Asn Ile His Thr Ala Ile Thr Gln Val Arg
Asn3345 3350 3355 3360Leu Ile Gly Lys Glu Glu Tyr Val Asp Tyr Met
Pro Val Met Lys Arg 3365 3370 3375 Tyr Ser Ala Pro Ser Glu Ser Glu
Gly Val Leu 3380 3385 203392PRTDengue 1 virus strain WP 20Met Asn
Asn Gln Arg Lys Lys Thr Gly Arg Pro Ser Phe Asn Met Leu 1 5 10 15
Lys Arg Ala Arg Asn Arg Val Ser Thr Val Ser Gln Leu Ala Lys Arg 20
25 30 Phe Ser Lys Gly Leu Leu Ser 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 Ile Leu Ala Arg Trp Gly Ser Phe Lys Lys Asn
Gly Ala Ile Lys Val65 70 75 80 Leu Arg Gly Phe Lys Lys Glu Ile Ser
Asn Met Leu Asn Ile Met Asn 85 90 95 Arg Arg Lys Arg Ser Val Thr
Met Leu Leu Met Leu Leu Pro Thr Ala 100 105 110 Leu Ala Phe His Leu
Thr Thr Arg Gly Gly Glu Pro His Met Ile Val 115 120 125 Ser Lys Gln
Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala Gly 130 135 140 Val
Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu145 150
155 160 Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Thr Glu Pro
Asp 165 170 175 Asp Val Asp Cys Trp Cys Asn Ala Thr Glu Thr Trp Val
Thr Tyr Gly 180 185 190 Thr Cys Ser Gln Thr Gly Glu His Arg Arg Asp
Lys Arg Ser Val Ala 195 200 205 Leu Ala Pro His Val Gly Leu Gly Leu
Glu Thr Arg Thr Glu Thr Trp 210 215 220 Met Ser Ser Glu Gly Ala Trp
Lys Gln Ile Gln Lys Val Glu Thr Trp225 230 235 240 Ala Leu Arg His
Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His 245 250 255 Ala Ile
Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu 260 265 270
Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile 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 Ser Cys Val Thr Thr Met Ala Lys Asp
Lys Pro Thr305 310 315 320 Leu Asp Ile Glu Leu Leu Lys Thr Glu Val
Thr Asn Pro Ala Val Leu 325 330 335 Arg Lys Leu Cys Ile Glu Ala Lys
Ile Ser Asn Thr Thr Thr Asp Ser 340 345 350 Arg Cys Pro Thr Gln Gly
Glu Ala Thr Leu Val Glu Glu Gln Asp Thr 355 360 365 Asn Phe Val Cys
Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn Gly 370 375 380 Cys Gly
Leu Phe Gly Lys Gly Ser Leu Ile Thr Cys Ala Lys Phe Lys385 390 395
400 Cys Val Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu Asn Leu Lys
405 410 415 Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His Gln
Val Gly 420 425 430 Asn Glu Thr Thr Glu His Gly Thr Thr Ala Thr Ile
Thr Pro Gln Ala 435 440 445 Pro Thr Ser Glu Ile Gln Leu Thr Asp Tyr
Gly Ala Leu Thr Leu Asp 450 455 460 Cys Ser Pro Arg Thr Gly Leu Asp
Phe Asn Glu Met Val Leu Leu Thr465 470 475 480 Met Glu Lys Lys Ser
Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu 485 490 495 Pro Leu Pro
Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn 500 505 510 Arg
Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln 515 520
525 Glu 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 Thr Ser Gly Thr Thr Thr Ile
Phe Ala545 550 555 560 Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys
Leu Thr Leu Lys Gly 565 570 575 Met Ser Tyr Val Met Cys Thr Gly Ser
Phe Lys Leu Glu Lys Glu Val 580 585 590 Ala Glu Thr Gln His Gly Thr
Val Leu Val Gln Val Lys Tyr Glu Gly 595 600 605 Thr Asp Ala Pro Cys
Lys Ile Pro Phe Ser Ser Gln Asp Glu Lys Gly 610 615 620 Val Thr Gln
Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp625 630 635 640
Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser 645
650 655 Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp
Phe 660 665 670 Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr
Ala Arg Gly 675 680 685 Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala
Trp Asp Phe Gly Ser 690 695 700 Ile Gly Gly Val Phe Thr Ser Val Gly
Lys Leu Ile His Gln Ile Phe705 710 715 720 Gly Thr Ala Tyr Gly Val
Leu Phe Ser Gly Val Ser Trp Thr Met Lys 725 730 735 Ile Gly Ile Gly
Ile Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Ser 740 745 750 Thr Ser
Leu Ser Met Thr Cys Ile Ala Val Gly Met Val Thr Leu Tyr 755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Ile Asn Trp Lys 770
775 780 Gly Arg Glu Leu Lys Cys Gly Ser Gly Ile Phe Val Thr Asn Glu
Val785 790 795 800 His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Ala Asp
Ser Pro Lys Arg 805 810 815 Leu Ser Ala Ala Ile Gly Lys Ala Trp Glu
Glu Gly Val Cys Gly Ile 820 825 830 Arg Ser Ala Thr Arg Leu Glu Asn
Ile Met Trp Lys Gln Ile Ser Asn 835 840 845 Glu Leu Asn His Ile Leu
Leu Glu Asn Asp Met Lys Phe Thr Val Val 850 855 860 Val Gly Asp Val
Ser Gly Ile Leu Ala Gln Gly Lys Lys Met Ile Arg865 870 875 880 Pro
Gln Pro Met Glu His Lys Tyr Ser Trp Lys Ser Trp Gly Lys Ala 885 890
895 Lys Ile Ile Gly Ala Asp Val Gln Asn Thr Thr Phe Ile Ile Asp Gly
900 905 910 Pro Asn Thr Pro Glu Cys Pro Asp Asn Gln Arg Ala Trp Asn
Ile Trp 915 920 925 Glu
Val Glu Asp Tyr Gly Phe Gly Ile Phe Thr Thr Asn Ile Trp Leu 930 935
940 Lys Leu Arg Asp Ser Tyr Thr Gln Val Cys Asp His Arg Leu Met
Ser945 950 955 960 Ala Ala Ile Lys Asp Ser Lys Ala Val His Ala Asp
Met Gly Tyr Trp 965 970 975 Ile Glu Ser Glu Lys Asn Glu Thr Trp Lys
Leu Ala Arg Ala Ser Phe 980 985 990 Ile Glu Val Lys Thr Cys Ile Trp
Pro Lys Ser His Thr Leu Trp Ser 995 1000 1005 Asn Gly Val Leu Glu
Ser Glu Met Ile Ile Pro Lys Ile Tyr Gly Gly 1010 1015 1020 Pro Ile
Ser Gln His Asn Tyr Arg Pro Gly Tyr Phe Thr Gln Thr Ala1025 1030
1035 1040Gly Pro Trp His Leu Gly Lys Leu Glu Leu Asp Phe Asp Leu
Cys Glu 1045 1050 1055 Gly Thr Thr Val Val Val Asp Glu His Cys Gly
Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Val Thr Gly
Lys Thr Ile His Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr Leu
Pro Pro Leu Arg Phe Lys Gly Glu Asp Gly Cys 1090 1095 1100 Trp Tyr
Gly Met Glu Ile Arg Pro Val Lys Glu Lys Glu Glu Asn Leu1105 1110
1115 1120Val Lys Ser Met Val Ser Ala Gly Ser Gly Glu Val Asp Ser
Phe Ser 1125 1130 1135 Leu Gly Leu Leu Cys Ile Ser Ile Met Ile Glu
Glu Val Met Arg Ser 1140 1145 1150 Arg Trp Ser Arg Lys Met Leu Met
Thr Gly Thr Leu Ala Val Phe Leu 1155 1160 1165 Leu Leu Thr Met Gly
Gln Leu Thr Trp Asn Asp Leu Ile Arg Leu Cys 1170 1175 1180 Ile Met
Val Gly Ala Asn Ala Ser Asp Lys Met Gly Met Gly Thr Thr1185 1190
1195 1200Tyr Leu Ala Leu Met Ala Thr Phe Arg Met Arg Pro Met Phe
Ala Val 1205 1210 1215 Gly Leu Leu Phe Arg Arg Leu Thr Ser Arg Glu
Val Leu Leu Leu Thr 1220 1225 1230 Val Gly Leu Ser Leu Val Ala Ser
Val Glu Leu Pro Asn Ser Leu Glu 1235 1240 1245 Glu Leu Gly Asp Gly
Leu Ala Met Gly Ile Met Met Leu Lys Leu Leu 1250 1255 1260 Thr Asp
Phe Gln Ser His Gln Leu Trp Ala Thr Leu Leu Ser Leu Thr1265 1270
1275 1280Phe Val Lys Thr Thr Phe Ser Leu His Tyr Ala Trp Lys Thr
Met Ala 1285 1290 1295 Met Ile Leu Ser Ile Val Ser Leu Phe Pro Leu
Cys Leu Ser Thr Thr 1300 1305 1310 Ser Gln Lys Thr Thr Trp Leu Pro
Val Leu Leu Gly Ser Leu Gly Cys 1315 1320 1325 Lys Pro Leu Thr Met
Phe Leu Ile Thr Glu Asn Lys Ile Trp Gly Arg 1330 1335 1340 Lys Ser
Trp Pro Leu Asn Glu Gly Ile Met Ala Val Gly Ile Val Ser1345 1350
1355 1360Ile Leu Leu Ser Ser Leu Leu Lys Asn Asp Val Pro Leu Ala
Gly Pro 1365 1370 1375 Leu Ile Ala Gly Gly Met Leu Ile Ala Cys Tyr
Val Ile Ser Gly Ser 1380 1385 1390 Ser Ala Asp Leu Ser Leu Glu Lys
Ala Ala Glu Val Ser Trp Glu Glu 1395 1400 1405 Glu Ala Glu His Ser
Gly Ala Ser His Asn Ile Leu Val Glu Val Gln 1410 1415 1420 Asp Asp
Gly Thr Met Lys Ile Lys Asp Glu Glu Arg Asp Asp Thr Leu1425 1430
1435 1440Thr Ile Leu Leu Lys Ala Thr Leu Leu Ala Ile Ser Gly Val
Tyr Pro 1445 1450 1455 Met Ser Ile Pro Ala Thr Leu Phe Val Trp Tyr
Phe Trp Gln Lys Lys 1460 1465 1470 Lys Gln Arg Ser Gly Val Leu Trp
Asp Thr Pro Ser Pro Pro Glu Val 1475 1480 1485 Glu Arg Ala Val Leu
Asp Asp Gly Ile Tyr Arg Ile Leu Gln Arg Gly 1490 1495 1500 Leu Leu
Gly Arg Ser Gln Val Gly Val Gly Val Phe Gln Glu Gly Val1505 1510
1515 1520Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met
Tyr Gln 1525 1530 1535 Gly Lys Arg Leu Glu Pro Ser Trp Ala Ser Val
Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Arg Phe
Gln Gly Ser Trp Asn Ala Gly Glu 1555 1560 1565 Glu Val Gln Val Ile
Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Val 1570 1575 1580 Gln Thr
Ala Pro Gly Thr Phe Lys Thr Pro Glu Gly Glu Val Gly Ala1585 1590
1595 1600Ile Ala Leu Asp Phe Lys Pro Gly Thr Ser Gly Ser Pro Ile
Val Asn 1605 1610 1615 Arg Glu Gly Lys Ile Val Gly Leu Tyr Gly Asn
Gly Val Val Thr Thr 1620 1625 1630 Ser Gly Thr Tyr Val Ser Ala Ile
Ala Gln Ala Lys Ala Ser Gln Glu 1635 1640 1645 Gly Pro Leu Pro Glu
Ile Glu Asp Glu Val Phe Arg Lys Arg Asn Leu 1650 1655 1660 Thr Ile
Met Asp Leu His Pro Gly Ser Gly Lys Thr Arg Arg Tyr Leu1665 1670
1675 1680Pro Ala Ile Val Arg Glu Ala Ile Arg Arg Asn Val Arg Thr
Leu Val 1685 1690 1695 Leu Ala Pro Thr Arg Val Val Ala Ser Glu Met
Ala Glu Ala Leu Lys 1700 1705 1710 Gly Met Pro Ile Arg Tyr Gln Thr
Thr Ala Val Lys Ser Glu His Thr 1715 1720 1725 Gly Lys Glu Ile Val
Asp Leu Met Cys His Ala Thr Phe Thr Met Arg 1730 1735 1740 Leu Leu
Ser Pro Val Arg Val Pro Asn Tyr Asn Met Ile Ile Met Asp1745 1750
1755 1760Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
Tyr Ile 1765 1770 1775 Ser Thr Arg Val Gly Met Gly Glu Ala Ala Ala
Ile Phe Met Thr Ala 1780 1785 1790 Thr Pro Pro Gly Ser Val Glu Ala
Phe Pro Gln Ser Asn Ala Val Ile 1795 1800 1805 Gln Asp Glu Glu Arg
Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Tyr 1810 1815 1820 Asp Trp
Ile Thr Asp Phe Pro Gly Lys Thr Val Trp Phe Val Pro Ser1825 1830
1835 1840Ile Lys Ser Gly Asn Asp Ile Ala Asn Cys Leu Arg Lys Asn
Gly Lys 1845 1850 1855 Arg Val Val Gln Leu Ser Arg Lys Thr Phe Asp
Thr Glu Tyr Gln Lys 1860 1865 1870 Thr Lys Asn Asn Asp Trp Asp Tyr
Val Val Thr Thr Asp Ile Ser Glu 1875 1880 1885 Met Gly Ala Asn Phe
Arg Ala Asp Arg Val Ile Asp Pro Arg Arg Cys 1890 1895 1900 Leu Lys
Pro Val Ile Leu Lys Asp Gly Pro Glu Arg Val Ile Leu Ala1905 1910
1915 1920Gly Pro Met Pro Val Thr Val Ala Ser Ala Ala Gln Arg Arg
Gly Arg 1925 1930 1935 Ile Gly Arg Asn Gln Asn Lys Glu Gly Asp Gln
Tyr Ile Tyr Met Gly 1940 1945 1950 Gln Pro Leu Asn Asn Asp Glu Asp
His Ala His Trp Thr Glu Ala Lys 1955 1960 1965 Met Leu Leu Asp Asn
Ile Asn Thr Pro Glu Gly Ile Ile Pro Ala Leu 1970 1975 1980 Phe Glu
Pro Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly Glu Tyr Arg1985 1990
1995 2000Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Glu Leu Met Arg
Arg Gly 2005 2010 2015 Asp Leu Pro Val Trp Leu Ser Tyr Lys Val Ala
Ser Glu Gly Phe Gln 2020 2025 2030 Tyr Ser Asp Arg Arg Trp Cys Phe
Asp Gly Glu Arg Asn Asn Gln Val 2035 2040 2045 Leu Glu Glu Asn Met
Asp Val Glu Ile Trp Thr Lys Glu Gly Glu Arg 2050 2055 2060 Lys Lys
Leu Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr Ser Asp Pro2065 2070
2075 2080Leu Ala Leu Arg Glu Phe Lys Glu Phe Ala Ala Gly Arg Arg
Ser Val 2085 2090 2095 Ser Gly Asp Leu Ile Leu Glu Ile Gly Lys Leu
Pro Gln His Leu Thr 2100 2105 2110 Gln Arg Ala Gln Asn Ala Leu Asp
Asn Leu Val Met Leu His Asn Ser 2115 2120 2125 Glu Gln Gly Gly Lys
Ala Tyr Arg His Ala Met Glu Glu Leu Pro Asp 2130 2135 2140 Thr Ile
Glu Thr Leu Met Leu Leu Ala Leu Ile Ala Val Leu Thr Gly2145 2150
2155 2160Gly Val Thr Leu Phe Phe Leu Ser Gly Arg Gly Leu Gly Lys
Thr Ser 2165 2170 2175 Ile Gly Leu Leu Cys Val Ile Ala Ser Ser Ala
Leu Leu Trp Met Ala 2180 2185 2190 Ser Val Glu Pro His Trp Ile Ala
Ala Ser Ile Ile Leu Glu Phe Phe 2195 2200 2205 Leu Met Val Leu Leu
Ile Pro Glu Pro Asp Arg Gln Arg Thr Pro Gln 2210 2215 2220 Asp Asn
Gln Leu Ala Tyr Val Val Ile Gly Leu Leu Phe Met Ile Leu2225 2230
2235 2240Thr Ala Ala Ala Asn Glu Met Gly Leu Leu Glu Thr Thr Lys
Lys Asp 2245 2250 2255 Leu Gly Ile Gly His Ala Ala Ala Glu Asn His
His His Ala Ala Met 2260 2265 2270 Leu Asp Val Asp Leu His Pro Ala
Ser Ala Trp Thr Leu Tyr Ala Val 2275 2280 2285 Ala Thr Thr Ile Ile
Thr Pro Met Met Arg His Thr Ile Glu Asn Thr 2290 2295 2300 Thr Ala
Asn Ile Ser Leu Thr Ala Ile Ala Asn Gln Ala Ala Ile Leu2305 2310
2315 2320Met Gly Leu Asp Lys Gly Trp Pro Ile Ser Lys Met Asp Ile
Gly Val 2325 2330 2335 Pro Leu Leu Ala Leu Gly Cys Tyr Ser Gln Val
Asn Pro Leu Thr Leu 2340 2345 2350 Thr Ala Ala Val Phe Met Leu Val
Ala His Tyr Ala Ile Ile Gly Pro 2355 2360 2365 Gly Leu Gln Ala Lys
Ala Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala 2370 2375 2380 Gly Ile
Met Lys Asn Pro Thr Val Asp Gly Ile Val Ala Ile Asp Leu2385 2390
2395 2400Asp Pro Val Val Tyr Asp Ala Lys Phe Glu Lys Gln Leu Gly
Gln Ile 2405 2410 2415 Met Leu Leu Ile Leu Cys Thr Ser Gln Ile Leu
Leu Met Arg Thr Thr 2420 2425 2430 Trp Ala Leu Cys Glu Ser Ile Thr
Leu Ala Thr Gly Pro Leu Thr Thr 2435 2440 2445 Leu Trp Glu Gly Ser
Pro Gly Lys Phe Trp Asn Thr Thr Ile Ala Val 2450 2455 2460 Ser Met
Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu2465 2470
2475 2480Ala Phe Ser Leu Met Lys Ser Leu Gly Gly Gly Arg Arg Gly
Thr Gly 2485 2490 2495 Ala Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys
Arg Gln Leu Asn Gln 2500 2505 2510 Leu Ser Lys Ser Glu Phe Asn Thr
Tyr Lys Arg Ser Gly Ile Ile Glu 2515 2520 2525 Val Asp Arg Ser Glu
Ala Lys Glu Gly Leu Lys Arg Gly Glu Pro Thr 2530 2535 2540 Lys His
Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp Phe Val Glu2545 2550
2555 2560Arg Asn Leu Val Lys Pro Glu Gly Lys Val Ile Asp Leu Gly
Cys Gly 2565 2570 2575 Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly Leu
Lys Lys Val Thr Glu 2580 2585 2590 Val Lys Gly Tyr Thr Lys Gly Gly
Pro Gly His Glu Glu Pro Ile Pro 2595 2600 2605 Met Ala Thr Tyr Gly
Trp Asn Leu Val Lys Leu Tyr Ser Gly Lys Asp 2610 2615 2620 Val Phe
Phe Thr Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile2625 2630
2635 2640Gly Glu Ser Ser Pro Asn Pro Thr Ile Glu Glu Gly Arg Thr
Leu Arg 2645 2650 2655 Val Leu Lys Met Val Glu Pro Trp Leu Arg Gly
Asn Gln Phe Cys Ile 2660 2665 2670 Lys Ile Leu Asn Pro Tyr Met Pro
Ser Val Val Glu Thr Leu Glu Gln 2675 2680 2685 Met Gln Arg Lys His
Gly Gly Met Leu Val Arg Asn Pro Leu Ser Arg 2690 2695 2700 Asn Ser
Thr His Glu Met Tyr Trp Val Ser Cys Gly Thr Gly Asn Ile2705 2710
2715 2720Val Ser Ala Val Asn Met Thr Ser Arg Met Leu Leu Asn Arg
Phe Thr 2725 2730 2735 Met Ala His Arg Lys Pro Thr Tyr Glu Arg Asp
Val Asp Leu Gly Ala 2740 2745 2750 Gly Thr Arg His Val Ala Val Glu
Pro Glu Val Ala Asn Leu Asp Ile 2755 2760 2765 Ile Gly Gln Arg Ile
Glu Asn Ile Lys Asn Gly His Lys Ser Thr Trp 2770 2775 2780 His Tyr
Asp Glu Asp Asn Pro Tyr Lys Thr Trp Ala Tyr His Gly Ser2785 2790
2795 2800Tyr Glu Val Lys Pro Ser Gly Ser Ala Ser Ser Met Val Asn
Gly Val 2805 2810 2815 Val Arg Leu Leu Thr Lys Pro Trp Asp Val Ile
Pro Met Val Thr Gln 2820 2825 2830 Ile Ala Met Thr Asp Thr Thr Pro
Phe Gly Gln Gln Arg Val Phe Lys 2835 2840 2845 Glu Lys Val Asp Thr
Arg Thr Pro Lys Ala Lys Arg Gly Thr Ala Gln 2850 2855 2860 Ile Met
Glu Val Thr Ala Arg Trp Leu Trp Gly Phe Leu Ser Arg Asn2865 2870
2875 2880Lys Lys Pro Arg Ile Cys Thr Arg Glu Glu Phe Thr Arg Lys
Val Arg 2885 2890 2895 Ser Asn Ala Ala Ile Gly Ala Val Phe Val Asp
Glu Asn Gln Trp Asn 2900 2905 2910 Ser Ala Lys Glu Ala Val Glu Asp
Glu Arg Phe Trp Asp Leu Val His 2915 2920 2925 Arg Glu Arg Glu Leu
His Lys Gln Gly Lys Cys Ala Thr Cys Val Tyr 2930 2935 2940 Asn Met
Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala2945 2950
2955 2960Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
Phe Leu 2965 2970 2975 Glu Phe Glu Ala Leu Gly Phe Met Asn Glu Asp
His Trp Phe Ser Arg 2980 2985 2990 Glu Asn Ser Leu Ser Gly Val Glu
Gly Glu Gly Leu His Lys Leu Gly 2995 3000 3005 Tyr Ile Leu Arg Asp
Ile Ser Lys Ile Pro Gly Gly Asn Met Tyr Ala 3010 3015 3020 Asp Asp
Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp Leu Gln3025 3030
3035 3040Asn Glu Ala Lys Ile Thr Asp Ile Met Glu Pro Glu His Ala
Leu Leu 3045 3050 3055 Ala Thr Ser Ile Phe Lys Leu Thr Tyr Gln Asn
Lys Val Val Arg Val 3060 3065 3070 Gln Arg Pro Ala Lys Asn Gly Thr
Val Met Asp Val Ile Ser Arg Arg 3075 3080 3085 Asp Gln Arg Gly Ser
Gly Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe 3090 3095 3100 Thr Asn
Met Glu Ala Gln Leu Ile Arg Gln Met Glu Ser Glu Gly Ile3105 3110
3115 3120Phe Ser Pro Ser Glu Leu Glu Thr Pro Asn Leu Ala Glu Arg
Val Leu 3125 3130 3135 Asp Trp Leu Lys Lys His Gly Thr Glu Arg Leu
Lys Arg Met Ala Ile 3140 3145 3150 Ser Gly Asp Asp Cys Val Val Lys
Pro Ile Asp Asp Arg Phe Ala Thr 3155 3160 3165 Ala Leu Thr Ala Leu
Asn Asp Met Gly Lys Val Arg Lys Asp Ile Pro 3170 3175 3180 Gln Trp
Glu Pro Ser Lys Gly Trp Asn Asp Trp Gln Gln Val Pro Phe3185 3190
3195 3200Cys Ser His His Phe His Gln Leu Ile Met Lys Asp Gly Arg
Glu Ile 3205 3210 3215 Val Val Pro Cys Arg Asn Gln Asp Glu Leu Val
Gly Arg Ala Arg Val 3220 3225 3230 Ser Gln Gly Ala Gly Trp Ser Leu
Arg Glu Thr Ala Cys Leu Gly Lys 3235 3240
3245 Ser Tyr Ala Gln Met Trp Gln Leu Met Tyr Phe His Arg Arg Asp
Leu 3250 3255 3260 Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro
Val Asp Trp Val3265 3270 3275 3280Pro Thr Ser Arg Thr Thr Trp Ser
Ile His Ala His His Gln Trp Met 3285 3290 3295 Thr Thr Glu Asp Met
Leu Ser Val Trp Asn Arg Val Trp Ile Glu Glu 3300 3305 3310 Asn Pro
Trp Met Glu Asp Lys Thr His Val Ser Ser Trp Glu Asp Val 3315 3320
3325 Pro Tyr Leu Gly Lys Arg Glu Asp Arg Trp Cys Gly Ser Leu Ile
Gly 3330 3335 3340 Leu Thr Ala Arg Ala Thr Trp Ala Thr Asn Ile Gln
Val Ala Ile Asn3345 3350 3355 3360Gln Val Arg Arg Leu Ile Gly Asn
Glu Asn Tyr Leu Asp Phe Met Thr 3365 3370 3375 Ser Met Lys Arg Phe
Lys Asn Glu Ser Asp Pro Glu Gly Ala Leu Trp 3380 3385 3390
213391PRTDengue 2 virus strain NGC 21Met 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
Val65 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 Ala Gly Met Ile Ile Met
Leu Ile 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 Glu Asp Gly 130 135 140 Val Asn Met Cys Thr
Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu145 150 155 160 Asp Thr
Ile Thr Tyr Lys Cys Pro Phe Leu Arg 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 Trp225 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 Ala Leu Ile Phe Ile Leu Leu 260 265 270 Thr Ala Val 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 Thr305
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 Ser 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 Thr385 390 395 400 Cys Lys Lys
Asn Met Lys Gly Lys Val Val Gln Pro Glu Asn Leu Glu 405 410 415 Tyr
Thr Ile 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 Ile 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 Gln465 470 475 480 Met Glu Asn 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 Thr545 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 Val 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 Glu625 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 Asn Trp Phe 660 665 670
Lys Lys Gly Ser Ser Ile Gly Gln Met Ile 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 Phe705 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 Val 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 Ile Thr Asp Asn Val785 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 Pro 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 Ala Gly Lys Arg Ser Leu Gln865 870 875 880 Pro Gln Pro Thr Glu
Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala 885 890 895 Lys Met Leu
Ser Thr Glu Ser 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 Lys Leu Arg Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu
Met Ser945 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 Ile Ile Pro Lys Asn Phe Ala Gly 1010 1015 1020 Pro
Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Thr Ala1025
1030 1035 1040Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
Phe Cys Glu 1045 1050 1055 Gly Thr Thr Val Val Val Thr Glu Asp Cys
Gly Asn Arg Gly Pro Ser 1060 1065 1070 Leu Arg Thr Thr Thr Ala Ser
Gly Lys Leu Ile Thr Glu Trp Cys Cys 1075 1080 1085 Arg Ser Cys Thr
Leu Pro Pro Leu Arg Tyr Arg Gly Glu Asp Gly Cys 1090 1095 1100 Trp
Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu Lys Glu Glu Asn Leu1105
1110 1115 1120Val Asn Ser Leu Val Thr Ala Gly His Gly Gln Ile Asp
Asn Phe Ser 1125 1130 1135 Leu Gly Val Leu Gly Met Ala Leu Phe Leu
Glu Glu Met Leu Arg Thr 1140 1145 1150 Arg Val Gly Thr Lys His Ala
Ile Leu Leu Val Ala Val Ser Phe Val 1155 1160 1165 Thr Leu Ile Thr
Gly Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met 1170 1175 1180 Val
Met Val Gly Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr1185
1190 1195 1200Tyr Leu Ala Leu Leu Ala Ala Phe Lys Val Arg Pro Thr
Phe Ala Ala 1205 1210 1215 Gly Leu Leu Leu Arg Lys Leu Thr Ser Lys
Glu Leu Met Met Thr Thr 1220 1225 1230 Ile Gly Ile Val Leu Leu Ser
Gln Ser Thr Ile Pro Glu Thr Ile Leu 1235 1240 1245 Glu Leu Thr Asp
Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val 1250 1255 1260 Arg
Lys Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu1265
1270 1275 1280Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
Val Ser Cys 1285 1290 1295 Thr Ile Leu Ala Val Val Ser Val Ser Pro
Leu Phe Leu Thr Ser Ser 1300 1305 1310 Gln Gln Lys Ala Asp Trp Ile
Pro Leu Ala Leu Thr Ile Lys Gly Leu 1315 1320 1325 Asn Pro Thr Ala
Ile Phe Leu Thr Thr Leu Ser Arg Thr Asn Lys Lys 1330 1335 1340 Arg
Ser Trp Pro Leu Asn Glu Ala Ile Met Ala Val Gly Met Val Ser1345
1350 1355 1360Ile Leu Ala Ser Ser Leu Leu Lys Asn Asp Ile Pro Met
Thr Gly Pro 1365 1370 1375 Leu Val Ala Gly Gly Leu Leu Thr Val Cys
Tyr Val Leu Thr Gly Arg 1380 1385 1390 Ser Ala Asp Leu Glu Leu Glu
Arg Ala Ala Asp Val Lys Trp Glu Asp 1395 1400 1405 Gln Ala Glu Ile
Ser Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser 1410 1415 1420 Glu
Asp Gly Ser Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu1425
1430 1435 1440Thr Ile Leu Ile Arg Thr Gly Leu Leu Val Ile Ser Gly
Leu Phe Pro 1445 1450 1455 Val Ser Ile Pro Ile Thr Ala Ala Ala Trp
Tyr Leu Trp Glu Val Lys 1460 1465 1470 Lys 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 Gly 1490 1495 1500 Ile
Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr1505
1510 1515 1520Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
Met His Lys 1525 1530 1535 Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp
Val Lys Lys Asp Leu Ile 1540 1545 1550 Ser Tyr Gly Gly Gly Trp Lys
Leu Glu Gly Glu Trp Lys Glu Gly Glu 1555 1560 1565 Glu Val Gln Val
Leu Ala Leu Glu Pro Gly Lys Asn Pro Arg Ala Val 1570 1575 1580 Gln
Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala Gly Thr Ile Gly Ala1585
1590 1595 1600Val Ser Leu Asp Phe Ser Pro Gly Thr Ser Gly Ser Pro
Ile Ile Asp 1605 1610 1615 Lys Lys Gly Lys Val Val Gly Leu Tyr Gly
Asn Gly Val Val Thr Arg 1620 1625 1630 Ser Gly Ala Tyr Val Ser Ala
Ile Ala Gln Thr Glu Lys Ser Ile Glu 1635 1640 1645 Asp Asn Pro Glu
Ile Glu Asp Asp Ile Phe Arg Lys Arg Lys Leu Thr 1650 1655 1660 Ile
Met Asp Leu His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro1665
1670 1675 1680Ala Ile Val Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr
Leu Ile Leu 1685 1690 1695 Ala Pro Thr Arg Val Val Ala Ala Glu Met
Glu Glu Ala Leu Arg Gly 1700 1705 1710 Leu 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 Leu 1730 1735 1740 Leu
Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu1745
1750 1755 1760Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
Tyr Ile Ser 1765 1770 1775 Thr Arg Val Glu Met Gly Glu Ala Ala Gly
Ile Phe Met Thr Ala Thr 1780 1785 1790 Pro Pro Gly Ser Arg Asp Pro
Phe Pro Gln Ser Asn Ala Pro Ile Met 1795 1800 1805 Asp Glu Glu Arg
Glu Ile Pro Glu Arg Ser Trp Ser Ser Gly His Glu 1810 1815 1820 Trp
Val Thr Asp Phe Lys Gly Lys Thr Val Trp Phe Val Pro Ser Ile1825
1830 1835 1840Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu Arg Lys Asn
Gly Lys Lys 1845 1850 1855 Val Ile Gln Leu Ser Arg Lys Thr Phe Asp
Ser Glu Tyr Val Lys Thr 1860 1865 1870 Arg Thr Asn Asp Trp Asp Phe
Val Val Thr Thr Asp Ile Ser Glu Met 1875 1880 1885 Gly Ala Asn Phe
Lys Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met 1890 1895 1900 Lys
Pro Val Ile Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly1905
1910 1915 1920Pro Met Pro Val Thr His Ser Ser Ala Ala Gln Arg Arg
Gly Arg Ile 1925 1930 1935 Gly Arg Asn Pro Lys Asn Glu Asn Asp Gln
Tyr Ile Tyr Met Gly Glu 1940 1945 1950 Pro 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 Met Phe 1970 1975 1980 Glu
Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu1985
1990 1995 2000Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
Arg Gly Asp 2005 2010 2015 Leu Pro Val Trp Leu Ala Tyr Arg Val Ala
Ala Glu Gly Ile Asn Tyr 2020 2025 2030 Ala Asp Arg Arg Trp Cys Phe
Asp Gly Ile Lys Asn Asn Gln Ile Leu 2035 2040 2045 Glu Glu Asn Val
Glu Val Glu Ile Trp Thr Lys Glu Gly Glu Arg Lys 2050 2055 2060 Lys
Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile Tyr Ser Asp Pro Leu2065
2070 2075 2080Thr Leu Lys Glu Phe Lys Glu Phe Ala Ala Gly Arg Lys
Ser Leu Thr 2085 2090 2095 Leu Asn Leu Ile Thr Glu Met Gly Arg Leu
Pro Thr Phe Met Thr Gln 2100 2105 2110 Lys Ala Arg Asp Ala Leu Asp
Asn Leu Ala Val Leu His Thr Ala Glu 2115 2120 2125 Ala Gly Gly Arg
Ala Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr 2130 2135 2140 Leu
Glu Thr Leu Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly2145
2150 2155 2160Ile Phe Leu Phe Leu
Met Ser Gly Arg Gly Ile Gly Lys Met Thr Leu 2165 2170 2175 Gly Met
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln 2180 2185
2190 Ile 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 Asp 2210 2215 2220 Asn Gln Leu Thr Tyr Val Val Ile Ala
Ile Leu Thr Val Val Ala Ala2225 2230 2235 2240Thr Met Ala Asn Glu
Met Gly Phe Leu Glu Lys Thr Lys Lys Asp Leu 2245 2250 2255 Gly Leu
Gly Ser Ile Thr Thr Gln Gln Pro Glu Ser Asn Ile Leu Asp 2260 2265
2270 Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala
Thr 2275 2280 2285 Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu
Asn Ser Ser Val 2290 2295 2300 Asn Val Ser Leu Thr Ala Ile Ala Asn
Gln Ala Thr Val Leu Met Gly2305 2310 2315 2320Leu Gly Lys Gly Trp
Pro Leu Ser Lys Met Asp Ile Gly Val Pro Leu 2325 2330 2335 Leu Ala
Ile Gly Cys Tyr Ser Gln Val Asn Pro Ile Thr Leu Thr Ala 2340 2345
2350 Ala Leu Phe Leu Leu Val Ala His Tyr Ala Ile Ile Gly Pro Gly
Leu 2355 2360 2365 Gln Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Ala
Ala Ala Gly Ile 2370 2375 2380 Met Lys Asn Pro Thr Val Asp Gly Ile
Thr Val Ile Asp Leu Asp Pro2385 2390 2395 2400Ile Pro Tyr Asp Pro
Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu 2405 2410 2415 Leu Val
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala 2420 2425
2430 Leu Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu
Trp 2435 2440 2445 Glu Gly Asn Pro Gly Arg 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 Leu Phe2465 2470 2475 2480Ser Ile Met Lys Asn
Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn Ile 2485 2490 2495 Gly Glu
Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala Leu Gly 2500 2505
2510 Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln Glu Val
Asp 2515 2520 2525 Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu
Thr Asp His His 2530 2535 2540 Ala Val Ser Arg Gly Ser Ala Lys Leu
Arg Trp Phe Val Glu Arg Asn2545 2550 2555 2560Met Val Thr Pro Glu
Gly Lys Val Val Asp Leu Gly Cys Gly Arg Gly 2565 2570 2575 Gly Trp
Ser Tyr Tyr Cys Gly Gly Leu Lys Asn Val Arg Glu Val Lys 2580 2585
2590 Gly Leu Thr Lys Gly Gly Pro Gly His Glu Glu Pro Ile Pro Met
Ser 2595 2600 2605 Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser Gly
Val Asp Val Phe 2610 2615 2620 Phe Thr Pro Pro Glu Lys Cys Asp Thr
Leu Leu Cys Asp Ile Gly Glu2625 2630 2635 2640Ser Ser Pro Asn Pro
Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu 2645 2650 2655 Asn Leu
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys 2660 2665
2670 Val Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala
Leu 2675 2680 2685 Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro
Leu Ser Arg Asn 2690 2695 2700 Ser Thr His Glu Met Tyr Trp Val Ser
Asn Ala Ser Gly Asn Ile Val2705 2710 2715 2720Ser Ser Val Asn Met
Ile Ser Arg Met Leu Ile Asn Arg Phe Thr Met 2725 2730 2735 Arg His
Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly Ser Gly 2740 2745
2750 Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu Asp Ile
Ile 2755 2760 2765 Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu
Thr Ser Trp His 2770 2775 2780 Tyr Asp Gln Asp His Pro Tyr Lys Thr
Trp Ala Tyr His Gly Ser Tyr2785 2790 2795 2800Glu Thr Lys Gln Thr
Gly Ser Ala Ser Ser Met Val Asn Gly Val Val 2805 2810 2815 Arg Leu
Leu Thr Lys Pro Trp Asp Val Val Pro Met Val Thr Gln Met 2820 2825
2830 Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys
Glu 2835 2840 2845 Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu Gly
Thr Lys Lys Leu 2850 2855 2860 Met Lys Ile Thr Ala Glu Trp Leu Trp
Lys Glu Leu Gly Lys Lys Lys2865 2870 2875 2880Thr Pro Arg Met Cys
Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser 2885 2890 2895 Asn Ala
Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser 2900 2905
2910 Ala 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 Asn 2930 2935 2940 Met Met Gly Lys Arg Glu Lys Lys Leu
Gly Glu Phe Gly Lys Ala Lys2945 2950 2955 2960Gly Ser Arg Ala Ile
Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu Glu 2965 2970 2975 Phe Glu
Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser Arg Glu 2980 2985
2990 Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly
Tyr 2995 3000 3005 Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala
Met Tyr Ala Asp 3010 3015 3020 Asp Thr Ala Gly Trp Asp Thr Arg Ile
Thr Leu Glu Asp Leu Lys Asn3025 3030 3035 3040Glu Glu Met Val Thr
Asn His Met Glu Gly Glu His Lys Lys Leu Ala 3045 3050 3055 Glu Ala
Ile Phe Lys Leu Thr Tyr Gln Asn Lys Val Val Arg Val Gln 3060 3065
3070 Arg Pro Thr Pro Arg Gly Thr Val Met Asp Ile Ile Ser Arg Arg
Asp 3075 3080 3085 Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gly Leu
Asn Thr Phe Thr 3090 3095 3100 Asn Met Glu Ala Gln Leu Ile Arg Gln
Met Glu Gly Glu Gly Val Phe3105 3110 3115 3120Lys Ser Ile Gln His
Leu Thr Val Thr Glu Glu Ile Ala Val Gln Asn 3125 3130 3135 Trp Leu
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser 3140 3145
3150 Gly Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser
Ala 3155 3160 3165 Leu Thr Ala Leu Asn Asp Met Gly Lys Val Arg Lys
Asp Ile Gln Gln 3170 3175 3180 Trp Glu Pro Ser Arg Gly Trp Asn Asp
Trp Thr Gln Val Pro Phe Cys3185 3190 3195 3200Ser His His Phe His
Glu Leu Ile Met Lys Asp Gly Arg Val Leu Val 3205 3210 3215 Val Pro
Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser 3220 3225
3230 Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys
Ser 3235 3240 3245 Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg
Arg Asp Leu Arg 3250 3255 3260 Leu Ala Ala Asn Ala Ile Cys Ser Ala
Val Pro Ser His Trp Val Pro3265 3270 3275 3280Thr Ser Arg Thr Thr
Trp Ser Ile His Ala Lys His Glu Trp Met Thr 3285 3290 3295 Thr Glu
Asp Met Leu Thr Val Trp Asn Arg Val Trp Ile Gln Glu Asn 3300 3305
3310 Pro Trp Met Glu Asp Lys Thr Pro Val Glu Ser Trp Glu Glu Ile
Pro 3315 3320 3325 Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser
Leu Ile Gly Leu 3330 3335 3340 Thr Ser Arg Ala Thr Trp Ala Lys Asn
Ile Gln Thr Ala Ile Asn Gln3345 3350 3355 3360Val Arg Ser Leu Ile
Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser 3365 3370 3375 Met Lys
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp 3380 3385 3390
223390PRTDengue 3 virus strain H87 22Met 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
Val65 70 75 80 Leu Lys Gly Phe Lys Lys Glu Ile Ser Asn Met Leu Ser
Ile Ile Asn 85 90 95 Lys 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 Asp145 150 155 160 Asp Thr
Val Thr Tyr Lys Cys Pro His 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 Trp225 230 235 240 Ala Leu 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 Thr305
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 Ile
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 Tyr 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 Gln385 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 Ser Gln Ala Ser Thr
435 440 445 Ala 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 Lys465 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 Lys Thr Pro Thr Trp Asn Arg 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 His545 550
555 560 Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Lys Leu Lys Gly Met
Ser 565 570 575 Tyr Ala Met Cys Leu Asn Thr 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 Glu625 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 Arg 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 Ser705 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
Ala Ile Gly Ile Ile Thr Leu Tyr Leu Gly 755 760 765 Val 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 Thr785 790 795
800 Trp Thr Glu Gln Tyr Lys Phe Gln Ala Asp Ser Pro Lys Arg Val 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 Asp Ile Lys
Leu Thr Val Val Val Gly 850 855 860 Asp Ile Thr Gly Val Leu Glu Gln
Gly Lys Arg Thr Leu Thr Pro Gln865 870 875 880 Pro Met Glu Leu Lys
Tyr Ser Trp Lys Thr Trp Gly Leu Ala Lys Ile 885 890 895 Val Thr Ala
Glu Thr Gln Asn Ser Ser Phe Ile Ile Asp Gly Pro Ser 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 Val Tyr Thr Gln Leu Cys Asp His Arg Leu Met Ser
Ala Ala945 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 Ile 1010 1015 1020 Ser
Gln His Asn His Arg Pro Gly Tyr His Thr Gln Thr Ala Gly Pro1025
1030 1035 1040Trp His Leu Gly Lys Leu Glu Leu Asp Phe Asn Tyr Cys
Glu Gly Thr 1045 1050 1055 Thr Val Val Ile Ser Glu Asn Cys Gly Thr
Arg Gly Pro Ser Leu Arg 1060 1065 1070 Thr Thr Thr Val Ser Gly Lys
Leu Ile His Glu Trp Cys Cys Arg Ser 1075
1080 1085 Cys Thr Leu Pro Pro Leu Arg Tyr Met Gly Glu Asp Gly Cys
Trp Tyr 1090 1095 1100 Gly Met Glu Ile Arg Pro Ile Asn Glu Lys Glu
Glu Asn Met Val Lys1105 1110 1115 1120Ser Leu Ala Ser Ala Gly Ser
Gly Lys Val Asp Asn Phe Thr Met Gly 1125 1130 1135 Val Leu Cys Leu
Ala Ile Leu Phe Glu Glu Val Met Arg Gly Lys Phe 1140 1145 1150 Gly
Lys Lys His Met Ile Ala Gly Val Leu Phe Thr Phe Val Leu Leu 1155
1160 1165 Leu Ser Gly Gln Ile Thr Trp Arg Gly Met Ala His Thr Leu
Ile Met 1170 1175 1180 Ile Gly Ser Asn Ala Ser Asp Arg Met Gly Met
Gly Val Thr Tyr Leu1185 1190 1195 1200Ala Leu Ile Ala Thr Phe Lys
Ile Gln Pro Phe Leu Ala Leu Gly Phe 1205 1210 1215 Phe Leu Arg Lys
Leu Thr Ser Arg Glu Asn Leu Leu Leu Gly Val Gly 1220 1225 1230 Leu
Ala Met Ala Ala Thr Leu Arg 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 Gln 1250 1255 1260 Phe Glu Thr Tyr Gln Leu Trp Thr Ala Leu Val
Ser Leu Thr Cys Ser1265 1270 1275 1280Asn Thr Ile Phe Thr Leu Thr
Val Ala Trp Arg Thr Ala Thr Leu Ile 1285 1290 1295 Leu Ala Gly Ile
Ser Leu Leu Pro Val Cys Gln Ser Ser Ser Met Arg 1300 1305 1310 Lys
Thr Asp Trp Leu Pro Met Thr Val Ala Ala Met Gly Val Pro Pro 1315
1320 1325 Leu Pro Leu Phe Ile Phe Ser Leu Lys Asp Thr Leu Lys Arg
Arg Ser 1330 1335 1340 Trp Pro Leu Asn Glu Gly Val Met Ala Val Gly
Leu Val Ser Ile Leu1345 1350 1355 1360Ala Ser Ser Leu Leu Arg Asn
Asp Val Pro Met Ala Gly Pro Leu Val 1365 1370 1375 Ala Gly Gly Leu
Leu Ile Ala Cys Tyr Val Ile Thr Gly Thr Ser Ala 1380 1385 1390 Asp
Leu Thr Val Glu Lys Ala Ala Asp Val Thr Trp Glu Glu Glu Ala 1395
1400 1405 Glu Gln Thr Gly Val Ser His Asn Leu Met Ile Thr Val Asp
Asp Asp 1410 1415 1420 Gly Thr Met Arg Ile Lys Asp Asp Glu Thr Glu
Asn Ile Leu Thr Val1425 1430 1435 1440Leu Leu Lys Thr Ala Leu Leu
Ile Val Ser Gly Ile Phe Pro Tyr Ser 1445 1450 1455 Ile Pro Ala Thr
Met Leu Val Trp His Thr Trp Gln Lys Gln Thr Gln 1460 1465 1470 Arg
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 Phe 1490 1495 1500 Gly Lys Thr Gln Val Gly Val Gly Val Gln Lys
Glu Gly Val Phe His1505 1510 1515 1520Thr Met Trp His Val Thr Arg
Gly Ala Val Leu Thr His Asn Gly Lys 1525 1530 1535 Arg Leu Glu Pro
Asn Trp Ala Ser Val Lys Lys Asp Leu Ile Ser Tyr 1540 1545 1550 Gly
Gly Gly Trp Arg Leu Ser Ala Gln Trp Gln Lys Gly Glu Glu Val 1555
1560 1565 Gln Val Ile Ala Val Glu Pro Gly Lys Asn Pro Lys Asn Phe
Gln Thr 1570 1575 1580 Met Pro Gly Ile Phe Gln Thr Thr Thr Gly Glu
Ile Gly Ala Ile Ala1585 1590 1595 1600Leu Asp Phe Lys Pro Gly Thr
Ser Gly Ser Pro Ile Ile Asn Arg Glu 1605 1610 1615 Gly Lys Val Val
Gly Leu Tyr Gly Asn Gly Val Val Thr Lys Asn Gly 1620 1625 1630 Gly
Tyr Val Ser Gly Ile Ala Gln Thr Asn Ala Glu Pro Asp Gly Pro 1635
1640 1645 Thr Pro Glu Leu Glu Glu Glu Met Phe Lys Lys Arg Asn Leu
Thr Ile 1650 1655 1660 Met Asp Leu His Pro Gly Ser Gly Lys Thr Arg
Lys Tyr Leu Pro Ala1665 1670 1675 1680Ile Val Arg Glu Ala Ile Lys
Arg Arg Leu Arg Thr Leu Ile Leu Ala 1685 1690 1695 Pro Thr Arg Val
Val Ala Ala Glu Met Glu Glu Ala Met Lys Gly Leu 1700 1705 1710 Pro
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 Leu 1730 1735 1740 Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile
Ile Met Asp Glu Ala1745 1750 1755 1760His Phe Thr Asp Pro Ala Ser
Ile Ala Ala Arg Gly Tyr Ile Ser Thr 1765 1770 1775 Arg Val Gly Met
Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro 1780 1785 1790 Pro
Gly Thr Ala Asp Ala Phe Pro Gln Ser Asn Ala Pro Ile Gln Asp 1795
1800 1805 Glu Glu Arg Asp Ile Pro Glu Arg Ser Trp Asn Ser Gly Asn
Glu Trp 1810 1815 1820 Ile Thr Asp Phe Val Gly Lys Thr Val Trp Phe
Val Pro Ser Ile Lys1825 1830 1835 1840Ala Gly Asn Val Ile Ala Asn
Cys Leu Arg Lys Asn Gly Lys Lys Val 1845 1850 1855 Ile Gln Leu Ser
Arg Lys Thr Phe Asp Thr Glu Tyr Gln Lys Thr Lys 1860 1865 1870 Leu
Asn Asp Trp Asp Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly 1875
1880 1885 Ala Asn Phe Ile Ala Asp Arg Val Ile Asp Pro Arg Arg Cys
Leu Lys 1890 1895 1900 Pro Val Ile Leu Thr Asp Gly Pro Glu Arg Val
Ile Leu Ala Gly Pro1905 1910 1915 1920Met Pro Val Thr Val Ala Ser
Ala Ala Gln Arg Arg Gly Arg Val Gly 1925 1930 1935 Arg Asn Pro Gln
Lys Glu Asn Asp Gln Tyr Ile Phe Met Gly Gln Pro 1940 1945 1950 Leu
Asn Lys 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 Glu 1970 1975 1980 Pro Glu Arg Glu Lys Ser Ala Ala Ile Asp Gly
Glu Tyr Arg Leu Lys1985 1990 1995 2000Gly Glu Ser Arg Lys Thr Phe
Val Glu Leu Met Arg Arg Gly Asp Leu 2005 2010 2015 Pro Val Trp Leu
Ala His Lys Val Ala Ser Glu Gly Ile Lys Tyr Thr 2020 2025 2030 Asp
Arg Lys Trp Cys Phe Asp Gly Glu Arg Asn Asn Gln Ile Leu Glu 2035
2040 2045 Glu Asn Met Asp Val Glu Ile Trp Thr Lys Glu Gly Glu Lys
Lys Lys 2050 2055 2060 Leu Arg Pro Arg Trp Leu Asp Ala Arg Thr Tyr
Ser Asp Pro Leu Ala2065 2070 2075 2080Leu Lys Glu Phe Lys Asp Phe
Ala Ala Gly Arg Lys Ser Ile Ala Leu 2085 2090 2095 Asp Leu Val Thr
Glu Ile Gly Arg Val Pro Ser His Leu Ala His Arg 2100 2105 2110 Thr
Arg Asn Ala Leu Asp Asn Leu Val Met Leu His Thr Ser Glu His 2115
2120 2125 Gly Gly Arg Ala Tyr Arg His Ala Val Glu Glu Leu Pro Glu
Thr Met 2130 2135 2140 Glu Thr Leu Leu Leu Leu Gly Leu Met Ile Leu
Leu Thr Gly Gly Ala2145 2150 2155 2160Met Leu Phe Leu Ile Ser Gly
Lys Gly Ile Gly Lys Thr Ser Ile Gly 2165 2170 2175 Leu Ile Cys Val
Ile Ala Ser Ser Gly Met Leu Trp Met Ala Asp Val 2180 2185 2190 Pro
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 Asn 2210 2215 2220 Gln Leu Ala Tyr Val Val Ile Gly Ile Leu Thr
Leu Ala Ala Ile Val2225 2230 2235 2240Ala Ala Asn Glu Met Gly Leu
Leu Glu Thr Thr Lys Arg Asp Leu Gly 2245 2250 2255 Met Ser Lys Glu
Pro Gly Val Val Ser Pro Thr Ser Tyr Leu Asp Val 2260 2265 2270 Asp
Leu His Pro Ala Ser Ala Trp Thr Leu Tyr Ala Val Ala Thr Thr 2275
2280 2285 Val Ile Thr Pro Met Leu Arg His Thr Ile Glu Asn Ser Thr
Ala Asn 2290 2295 2300 Val Ser Leu Ala Ala Ile Ala Asn Gln Ala Val
Val Leu Met Gly Leu2305 2310 2315 2320Asp Lys Gly Trp Pro Ile Ser
Lys Met Asp Leu Gly Val Pro Leu Leu 2325 2330 2335 Ala Leu Gly Cys
Tyr Ser Gln Val Asn Pro Leu Thr Leu Ile Ala Ala 2340 2345 2350 Val
Leu Leu Leu Val Thr His Tyr Ala Ile Ile Gly Pro Gly Leu Gln 2355
2360 2365 Ala Lys Ala Thr Arg Glu Ala Gln Lys Arg Thr Ala Ala Gly
Ile Met 2370 2375 2380 Lys Asn Pro Thr Val Asp Gly Ile Met Thr Ile
Asp Leu Asp Pro Val2385 2390 2395 2400Ile Tyr Asp Ser Lys Phe Glu
Lys Gln Leu Gly Gln Val Met Leu Leu 2405 2410 2415 Val Leu Cys Ala
Val Gln Leu Leu Leu Met Arg Thr Ser Trp Ala Leu 2420 2425 2430 Cys
Glu Val 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 Ala 2450 2455 2460 Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala
Gly Leu Ala Leu Ser2465 2470 2475 2480Ile Met Lys Ser Val Gly Thr
Gly Lys Arg Gly Thr Gly Ser Gln Gly 2485 2490 2495 Glu Thr Leu Gly
Glu Lys Trp Lys Lys Lys Leu Asn Gln Leu Ser Arg 2500 2505 2510 Lys
Glu Phe Asp Leu Tyr Lys Lys Ser Gly Ile Thr Glu Val Asp Arg 2515
2520 2525 Thr Glu Ala Lys Glu Gly Leu Lys Arg Gly Glu Ile Thr His
His Ala 2530 2535 2540 Val Ser Arg Gly Ser Ala Lys Leu Gln Trp Phe
Val Glu Arg Asn Met2545 2550 2555 2560Val Ile Pro Glu Gly Arg Val
Ile Asp Leu Gly Cys Gly Arg Gly Gly 2565 2570 2575 Trp Ser Tyr Tyr
Cys Ala Gly Leu Lys Lys Val Thr Glu Val Arg Gly 2580 2585 2590 Tyr
Thr Lys Gly Gly Pro Gly His Glu Glu Pro Val Pro Met Ser Thr 2595
2600 2605 Tyr Gly Trp Asn Ile Val Lys Leu Met Ser Gly Lys Asp Val
Phe Tyr 2610 2615 2620 Leu Pro Pro Glu Lys Cys Asp Thr Leu Leu Cys
Asp Ile Gly Glu Ser2625 2630 2635 2640Ser Pro Ser Pro Thr Val Glu
Glu Ser Arg Thr Ile Arg Val Leu Lys 2645 2650 2655 Met Val Glu Pro
Trp Leu Lys Asn Asn Gln Phe Cys Ile Lys Val Leu 2660 2665 2670 Asn
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 Thr 2690 2695 2700 His Glu Met Tyr Trp Ile Ser Asn Gly Thr Gly
Asn Ile Val Ser Ser2705 2710 2715 2720Val Asn Met Val Ser Arg Leu
Leu Leu Asn Arg Phe Thr Met Thr His 2725 2730 2735 Arg Arg Pro Thr
Ile Glu Lys Asp Val Asp Leu Gly Ala Gly Thr Arg 2740 2745 2750 His
Val Asn Ala Glu Pro Glu Thr Pro Asn Met Asp Val Ile Gly Glu 2755
2760 2765 Arg Ile Lys Arg Ile Lys Glu Glu His Ser Ser Thr Trp His
Tyr Asp 2770 2775 2780 Asp Glu Asn Pro Tyr Lys Thr Trp Ala Tyr His
Gly Ser Tyr Glu Val2785 2790 2795 2800Lys Ala Thr Gly Ser Ala Ser
Ser Met Ile Asn Gly Val Val Lys Leu 2805 2810 2815 Leu Thr Lys Pro
Trp Asp Val Val Pro Met Val Thr Gln Met Ala Met 2820 2825 2830 Thr
Asp Thr Thr Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val 2835
2840 2845 Asp Thr Arg Thr Pro Arg Pro Met Pro Gly Thr Arg Lys Val
Met Glu 2850 2855 2860 Ile Thr Ala Glu Trp Leu Trp Arg Thr Leu Gly
Arg Asn Lys Arg Pro2865 2870 2875 2880Arg Leu Cys Thr Arg Glu Glu
Phe Thr Lys Lys Val Arg Thr Asn Ala 2885 2890 2895 Ala Met Gly Ala
Val Phe Thr Glu Glu Asn Gln Trp Asp Ser Ala Arg 2900 2905 2910 Ala
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 Met 2930 2935 2940 Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly
Lys Ala Lys Gly Ser2945 2950 2955 2960Arg Ala Ile Trp Tyr Met Trp
Leu Gly Ala Arg Tyr Leu Glu Phe Glu 2965 2970 2975 Ala Leu Gly Phe
Leu Asn Glu Asp His Trp Phe Ser Arg Glu Asn Ser 2980 2985 2990 Tyr
Ser Gly Val Glu Gly Glu Gly Leu His Lys Leu Gly Tyr Ile Leu 2995
3000 3005 Arg Asp Ile Ser Lys Ile Pro Gly Gly Ala Met Tyr Ala Asp
Asp Thr 3010 3015 3020 Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp
Leu His Asn Glu Glu3025 3030 3035 3040Lys Ile Thr Gln Gln Met Asp
Pro Glu His Arg Gln Leu Ala Asn Ala 3045 3050 3055 Ile Phe Lys Leu
Thr Tyr Gln Asn Lys Val Val Lys Val Gln Arg Pro 3060 3065 3070 Thr
Pro Lys Gly Thr Val Met Asp Ile Ile Ser Arg Lys Asp Gln Arg 3075
3080 3085 Gly Ser Gly Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr
Asn Met 3090 3095 3100 Glu Ala Gln Leu Ile Arg Gln Met Glu Gly Glu
Gly Val Leu Ser Lys3105 3110 3115 3120Ala Asp Leu Glu Asn Pro His
Pro Leu Glu Lys Lys Ile Thr Gln Trp 3125 3130 3135 Leu Glu Thr Lys
Gly Val Glu Arg Leu Lys Arg Met Ala Ile Ser Gly 3140 3145 3150 Asp
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 Trp 3170 3175 3180 Gln Pro Ser Lys Gly Trp His Asp Trp Gln Gln
Val Pro Phe Cys Ser3185 3190 3195 3200His His Phe His Glu Leu Ile
Met Lys Asp Gly Arg Lys Leu Val Val 3205 3210 3215 Pro Cys Arg Pro
Gln Asp Glu Leu Ile Gly Arg Ala Arg Ile Ser Gln 3220 3225 3230 Gly
Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu Gly Lys Ala Tyr 3235
3240 3245 Ala Gln Met Trp Thr Leu Met Tyr Phe His Arg Arg Asp Leu
Arg Leu 3250 3255 3260 Ala Ser Asn Ala Ile Cys Ser Ala Val Pro Val
His Trp Val Pro Thr3265 3270 3275 3280Ser Arg Thr Thr Trp Ser Ile
His Ala His His Gln Trp Met Thr Thr 3285 3290 3295 Glu Asp Met Leu
Thr Val Trp Asn Arg Val Trp Ile Glu Asp Asn Pro 3300 3305 3310 Trp
Met Glu Asp Lys Thr Pro Val Thr Thr Trp Glu Asp Val Pro Tyr 3315
3320 3325 Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly
Leu Thr 3330 3335 3340 Ser Arg Ala Thr Trp Ala Gln Asn Ile Leu Thr
Ala Ile Gln Gln Val3345 3350 3355 3360Arg Ser Leu Ile Gly Asn Glu
Glu Phe Leu Asp Tyr Met Pro Ser Met 3365 3370 3375 Lys Arg Phe Arg
Lys Glu Glu Glu Ser Glu Gly Ala Ile Trp 3380 3385
33902321DNAArtificial SequenceOligonucleotide 23cagttccaaa
ccggaagctt g
212427DNAArtificial SequenceOligonucleotide 24ccaacgagct atcgtacgtt
ctctggg 272527DNAArtificial SequenceOligonucleotide 25gattgtgacc
atggcggccc atctttg 272631DNAArtificial SequenceOligonucleotide
26ggagattagg ccgctgagcg gtaaagaaga g 312726DNAArtificial
SequenceOligonucleotide 27gtttgtggaa aaatgtctga ggagaa
262827DNAArtificial SequenceOligonucleotide 28ctaggaaaca cataatatta
gttgtgg 272927DNAArtificial SequenceOligonucleotide 29cagatccacc
taaccataat ggcagtg 273025DNAArtificial SequenceOligonucleotide
30ggaaactcac ctcgggagag acagc 253126DNAArtificial
SequenceOligonucleotide 31ttgggtagag gtcaccgcac tcatcc
263225DNAArtificial SequenceOligonucleotide 32gtagaaatag ccgctctcat
cctag 253330DNAArtificial SequenceOligonucleotide 33ggcggcttac
gtaatgggag gtagctcagc 303427DNAArtificial SequenceOligonucleotide
34ctagagaagg cagcttctgt gcagtgg 273525DNAArtificial
SequenceOligonucleotide 35ccttggccat tccagcaaca atgac
253626DNAArtificial SequenceOligonucleotide 36gacgttcaaa ttttagccat
agaacc 263727DNAArtificial SequenceOligonucleotide 37ctggagaaac
gggcgccgta acattag 273827DNAArtificial SequenceOligonucleotide
38gaaattggat cggtaacctt agatttc 273927DNAArtificial
SequenceOligonucleotide 39ggagcagtaa cgtttgattt caaaccc
274024DNAArtificial SequenceOligonucleotide 40gttaccaaac ctggggatta
cgtc 244125DNAArtificial SequenceOligonucleotide 41gattaactat
catgaactta caccc 254225DNAArtificial SequenceOligonucleotide
42ggaaaacctt tggcaccgag tatcc 254326DNAArtificial
SequenceOligonucleotide 43tccagtgata ccggctagcg ctgctc
264423DNAArtificial SequenceOligonucleotide 44gcctcagagg tggccaaagg
aag 234526DNAArtificial SequenceOligonucleotide 45acatggaggc
agagatctgg actaga 264623DNAArtificial SequenceOligonucleotide
46aaagcatggc caaggatgct gtc 234728DNAArtificial
SequenceOligonucleotide 47gcataatgga cgctaagcat gactaagg
284826DNAArtificial SequenceOligonucleotide 48ttattgcata gtgcacgaaa
agcatg 264923DNAArtificial SequenceOligonucleotide 49gggcctatta
ttacgtaatg gac 235024DNAArtificial SequenceOligonucleotide
50ctgcaatcct ggtgatatta ttgc 245123DNAArtificial
SequenceOligonucleotide 51ctcataaaga acgttcaaac cct
235224DNAArtificial SequenceOligonucleotide 52cattagacag acgcgagttt
gaag 245327DNAArtificial SequenceOligonucleotide 53tggcgacgct
caagatagtg actgaag 275425DNAArtificial SequenceOligonucleotide
54gagtcatcat cgataccaac aatag 255527DNAArtificial
SequenceOligonucleotide 55cttcaaaacc tggcttctgc atcaaag
275628DNAArtificial SequenceOligonucleotide 56caaagatgtt gagcaacagg
ttcacaac 285727DNAArtificial SequenceOligonucleotide 57ggaaagaaga
aacacccgag actgtgc 275825DNAArtificial SequenceOligonucleotide
58gggaactggt cgatcgagaa agggc 255928DNAArtificial
SequenceOligonucleotide 59ccagtggatt actacagaag atatgctc
286029DNAArtificial SequenceOligonucleotide 60caggaacctg accggtaaag
aggaatacg 296128DNAArtificial SequenceOligonucleotide 61ctgtaattac
caacatcaaa caccaaag 286226DNAArtificial SequenceOligonucleotide
62ccaacaacaa ccaccaaagg ctattg 266325DNAArtificial
SequenceOligonucleotide 63ggattggtgt tgtcgatcca acagg
256424DNAArtificial SequencePrimer 64ctggtggaag cccaacacaa aaac
246545DNAArtificial SequencePrimer 65ctggtggaag gaagagagaa
attggcaact ccccaacaca aaaac 456622DNAArtificial SequencePrimer
66agaccccccc aagcatattg ac 226739DNAArtificial SequencePrimer
67agaccccccc aatatttcct cctcctatag catattgac 396821DNAArtificial
SequencePrimer 68cccaacacaa agcatattga c 21696DNAArtificial
SequenceOligonucleotide 69gcagcn 6705DNAArtificial
SequenceOligonucleotide 70gcagc 5
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