Supporting Information
Supplemental Table 1A. Summary of primers used in clone construction (fix primer
DEN7KB-).
primer
DEN#1
DEN2kbDEN2kb+
DENBGL4DENBLG3+
DEN7kbDEN5kb+
BsmbIDen
D:10198D:10166+
EGENEEGENE+
Dengue01+
Denv900-
direction
sense
antisense
sense
antisense
sense
antisense
sense
antisense
antisense
sense
antisense
sense
sense
antisense
positions
(-)31-21
2035-2014
2029-2051
3161-3136
3148-3176
7036-7013
5100-5120
10718-10684
10199-10166
10166-10199
2406-2393
2401-2424
422-442
901-871
sequence (5'-3')
NNNNNNGCGGCCGCTAATACGACTCACTATAGAGTTGTTAGTCTACGTGGACC
NNNNNCGTCTCGCCTCAATATTGACAGGCTCC
NNNNNNCGTCTCTGAGGCTGAACCTCCTTTTGGG
GCCAGCGAGGCTCTTTGGGATGATC
GAGCCTCGCTGGCCCTATTTCGCAACAC
TGCCTGGTTGGCTATAGCTGCCAG
CTGGGTCAGGAAAGACACGG
CGTCTCNNNNNAGAACCTGTTGATTCAACAGCACC
CTCTTCATTCCCTATAAGACTTCTCACCTGTTG
CAACAGGTGAGAAGTCTTATAGGGAATGAAGAG
NNNNNNGGTCTCACCACAGCTCCCAGATAGAGTG
NNNNNNGGTCTCTGTGGTACAAGCTGACATGGGGTG
CCAGCAGCACTTGCTTTCCAC
NNNNNNAACCACCTTCTGGGTCAAGGAAGTG
purpose
5' A fragment T7 promoter
3' A fragment A/B BsmbI site
5' B fragment A/B BsmBI site
3' B fragment B/C BglI site
5' C fragment B/C BglI site
3' C fragment C/D BglI site
5' D fragment C/D BglI site
3' D fragment BsmbI site @10707
3' DEN#1 K/O BglI site @10186
5' DEN#2 K/O BglI site @10186
3' E Gene/B fragment BsaI site
B fragment BsaI site
5' genotype I prM
3' genotype I prM
Position is nucleotide position on UNC 3001 (Ascession # pending). Native DENV sense
or anti-sense sequence shown in red except for D:10198- and D10166+ where red
indicates mutated nucleotides. N indicates random nucleotide. (-) and (+) indicate
positions extending beyond the 5’ (-) and 3’ (+) ends.
Supplemental Table 1B. Location and overhangs of ligation productions.
Junction
A/B
B/C
C/D
A/E Gene
E Gene/B
position
2040.00
3155.00
7030.00
933
2401
overhang-3'
GAGG-3'
GCU-3'
CCA-3'
CACTG-5'
CACC-5'
Enzyme
BsmbI
BglI
BglI
BstEII
BsaI
introduced
introduced
native
native
introduced
Junction refers to clone fragments. Overhangs are DNA sequence for the bolded
fragment under Junction.
Supplemental Table 2. FRNT50 values for human sera against the all four DENV
serotypes.
Serum
Infecting Serotype
Location
Year infected
Year collected
DENV-1
DENV-2
DENV-3
DENV-4
003*
DENV-3
Thailand
2001
2005
<1:10
<1:10
1:80
<1:10
005*
DENV-3
Puerto Rico
2000
2005
<1:10
<1:10
1:20
<1:10
011*
DENV-3
El Salvador
1998
2005
< 1:10
< 1:10
1:108
< 1:10
033*
103
DENV-3 DENV-3
India
Nicaragua
2005
1995
2009
2009
1:16
<1:20
1:11
<1:20
1:426
1:278
1:25
<1:20
105
DENV-3
Thailand
2002
2009
<1:20
<1:20
1:210
<1:20
109
DENV-3
S. Asia
ND
2010
<1:20
<1:20
1:231
<1:20
118
DENV-3
Nicaragua
2008
2010
1:60
1:32
1:980
1:60
009**
Secondary
India and Sri Lanka
2000
2005
1:160
>1:320
>1:320
1:320
006
DENV-1
endemic areas
1992
2005
1:866
1:30
1:37
<1:10
031**
DENV-2
South Pacific
1997
2005
1:20
>1:320
1:40
1:40
102
DENV-4
Honduras
2007
2009
<1:20
<1:20
<1:20
1:422
Infecting serotype was previously determined by neutralization assay against reference
WHO strains DENV1 WestPac-74, DENV2 S-16803, DENV3 CH-53489, and DENV4
TVP-360. Location and year refer to where and when the traveler acquired the DENV
infection. Infecting serotype was determined by the neutralization profile for individual
sera against all four serotypes. Fifty percent neutralization was determined by FACS
assay at UNC except where indicated.
*PRNT60 performed at NIH
**PRNT90 performed at CDC San Juan
Supplemental Table 3A. Summary of FRNT50 values for clones against homotypic
human polyclonal sera.
Serum
Infecting Serotype
Exposure & year
Indonesia '82 (I)
Thailand '95 (II)
SL '89 (III)
Cuba '02 (III)
PR '77 (IV)
003
DENV-3
Thailand '01
1:203
1:169
1:79
1:59
1:59
005
011
DENV-3
DENV-3
Puerto Rico '00 El Salvador '98
1:57
1:133
1:118
1:1091
1:118
1:1172
1:48
1:701
1:32
1:157
033
DENV-3
India '05
1:780
1:1269
1:1519
1:1256
1:304
103
DENV-3
Nicaragua '95
1:73
1:117
1:66
1:62
1:43
FRNT50 vaues are reported as a mean fold-dilution.
105
DENV-3
Thailand '02
1:139
1:349
1:118
1:69
1:44
109
DENV-3
South Asia
1:177
1:280
1:40
1:15
1:83
118
009
DENV-3
Secondary
Nicaragua '08 Endemic areas
1:533
1:544
1:1244
1:298
1:1013
1:340
1:623
1:175
1:207
1:250
Supplemental table 3B. Summary of FRNT50 values for clones against heterotypic
polyclonal sera.
Serum
001
006
102
Infecting Serotype
DENV-2
DENV-1
DENV-4
Exposure & year Sri Lanka 1996 Endemic areas South Pac '97
Indonesia '82 (I)
1:78
1:54
1:25
Thailand '95 (II)
1:17
1:11
1:58
SL '89 (III)
1:11
1:30
1:16
Cuba '02 (III)
1:40
1:17
1:12
PR '77 (IV)
1:25
1:7
1:29
FRNT50 values are reported as a mean fold-dilution
Supplemental Figure 1.
homotypic primary sera
5
Log(FRNT50) exp. 2
4
3
2
1
0
0
1
2
3
4
5
Log(FRNT50) exp. 1
Correlation between independent neutralization assays for homotypic sera against the
clone panel. Each point plots the first experiment FRNT50 (X axis) vs. second
experiment FRNT50 (Y axis) for a given serum sample against one clone. First and
second experiment FRNT50 values were highly correlated. N=28, R2=0.88, P<0.0001.
Dotted line shows 95% confidence interval for regression line.
Supplemental figure 2.
heterotypic primary sera
5
Log(FRNT50) exp. 2
4
3
2
1
0
0
1
2
3
4
5
Log(FRNT50) exp. 1
Correlation between independent neutralization assays for homotypic sera against the
clone panel. Each point plots the first experiment FRNT50 (X axis) vs. second
experiment FRNT50 (Y axis) for a given serum sample against one clone. There was no
correlation between first and second experiment FRNT50. N=10, R2=0.012, P=0.7596.
Dotted line shows 95% confidence interval for regression line.
Supplemental Figure 3.
Supplemental Figure 3. Summary of DENV-3 E gene informative amino acid positions
and domains. The table summarizes sites of amino acid variation are shown by domain
and position within the E protein, indicated as headers across the top of the table. Colors
represent differences at each position, with yellow indicating one difference and red
indicating the second difference per column. Variable sites that were predicted to be
exposed on the envelope protein structural surface were mapped onto a linear schematic
of the E protein to demonstrate which amino acids are proximal to one another and form
clusters that may represent important epitopes. Clusters are represented by color,
alphabetical lettering, and amino acid positions that occur within the cluster. Each cluster
color is noted on the linear E protein diagram by a similarly colored lollipop. Surface
exposed amino acid variation was mapped onto the structural model of the DENV3 E
protein, demonstrating that amino acids in all three domains change between different
genotypes. Domain I, orange; Domain II, Yellow; Domain III, blue. Surface exposed
variable residues occurring in: Domain I, red; Domain II, purple; and Domain III, cyan.