2
Supplementary Tables
Supplementary Table 1. ZIKV NS1-specific antibody measured by enzyme-linked immunosorbent assay (ELISA)
NHP ID Primary infection
IgM1 IgG1
Day 1 Day 3 Day 7 Day 1 Day 3 Day 7
5305 ND ND 0.153 ND ND 0.110 5687 ND ND 0.121 ND 0.548 1.683 5831 ND ND 0.125 0.092 0.092 1.378 5825 ND ND 0.142 ND ND ND 5169 ND 0.051 0.183 ND ND ND 5690 0.063 0.069 0.132 ND ND ND 5818 0.053 0.051 0.267 ND ND ND 5829 ND 0.051 0.404 0.073 ND 0.082 5939 ND ND 0.085 ND ND ND 5963 ND ND 0.077 ND ND ND
1Mean optical density (405 nm) is shown.
ND: not detected
Supplementary Table 2. Neutralizing antibody titers measured by 90% plaque reduction neutralization test (PRNT90)
NHP ID Primary infection Re-challenge
Day 7 Day 28 1Day 0 Day 7 5169 2 160 160 320 5690 2 320 1280 1280 5818 20 1280 320 1280 5829 20 160 80 80 5939 40 1280 640 1280 5963 20 160 160 640
1Re-challenge (Day 0) is equivalent to Day 45 post primary infection
Nature Medicine: doi:10.1038/nm.4206
3
Supplementary Figure Legends
Supplementary Figure 1. Viral dynamics and shedding in rhesus macaques infected with a
Puerto Rican (PR) isolate. ZIKV RNA was extracted from each specimen and quantitated by
qRT-PCR in (a) plasma, (b) urine, (c) saliva and (d) CSF.
Supplementary Figure 2. Blood chemistries during primary ZIKV infection. Blood
chemistries were analyzed on days 0, 3, 7, and 10 during primary ZIKV infection for male (blue,
n=5) and female (red, n=5) rhesus monkeys. Black line indicates median.
Supplementary Figure 3. Body temperature during primary ZIKV infection. Body
temperature was monitored via subcutaneously implanted transponders that recorded peripheral
temperature every 30 minutes during the first 14 days following ZIKV infection. Shown are the
mean temperatures for male (blue, n=5) and female (red, n=5) monkeys. Temperature fluctuations
on days 7 and 14 occurred during anesthesia for biopsies. Mean trough on day of infection
indicated by dashed line. Trough temperatures (averaged within the shaded gray regions) on each
day post infection were compared to those on day 0 using a repeated measured ANOVA followed
by a Dunnett's multiple comparison. Days 1 through 8 were significantly different from baseline
(day 0): Days 1 and 2 p < 0.01, days 3-7 p < 0.001, and day 8 p < 0.05.
Supplementary Figure 4. Complete blood counts during ZIKV infection. Compete blood
counts were analyzed on a) days 0, 1, 2, 3, 4, 5, 7, 10, and 14 during primary infection for 5 male
(blue) and 5 female (red) rhesus monkeys and on b) days 0, 1, 2, 3, 4, 5, and 7 during re-infection
for 3 male (blue) and 3 female (red) rhesus monkeys. Black line indicates median.
Nature Medicine: doi:10.1038/nm.4206
4
Supplementary Figure 5. Peripheral blood mononuclear cell activation and frequencies
during primary ZIKV infection. Activation and absolute numbers of T cells, B cells, NK cells
and monocytes were measured by flow cytometry on days 0, 1, 2, 3, 4, 5, 7, 10, and 14 following
ZIKV infection of 5 male and 5 female monkeys infected with a Thai ZIKV isolate. (a) Percent
activated (CD16+) and total numbers per µL whole blood of monocytes. Black line indicates
median. Individual values are shown for males (blue) and females (red). (b) Comparison of
percent CD16+ monocytes on day 2 of infection between males and females by a Mann-Whitney
test. Lines indicate median and interquartile range. Percent-activated (CD69+) and total numbers
per µL whole blood of naïve (CD28+CD95-), central memory (CD28+CD95+) and
effector/effector memory (CD28-CD95+) CD4+ (c) and CD8+ (d) T cells. (e) Percent activated
(CD69+) and total numbers per µL whole blood of CD16+, CD16-CD56+, and CD16-CD56- NK
cells. (f) CD38 expression (geometric mean fluorescence of CD38) and total numbers per µL
whole blood of naïve (CD27-) and memory (CD27+) B cells.
Supplementary Figure 6. ZIKV-specific T cells during primary ZIKV infection.
Degranulation and cytokine production of T cells in PBMC (a) and lymph nodes (b) were
measured by flow cytometry following stimulation of cells from days 0, 7, 14, 21 (PBMC only),
and 28 with overlapping peptide pools covering the entire capsid and envelope proteins of ZIKV.
Percent of cells expressing CD107a, IFNγ, TNFα, and IL-2 after a 6hr peptide stimulation is
shown. A threshold for determining positive values, after background subtraction of un-stimulated
corresponding samples, was applied as previously described [18]. Values below this threshold
were set to 0. Values greater than 1.5-fold above day of challenge were considered anamnestic.
Each monkey indicated by a different color. Triangles are males, circles females.
Nature Medicine: doi:10.1038/nm.4206
5
Supplementary Figure 7. Cytokine modulation in rhesus macaques following ZIKV
infection. The levels of various cytokines were measured in plasma on days 0, 1, 3, and 7 post
infection. (a) Heatmap of cytokine levels at 1, 3, and 7 days post-infection (dpi). Each cytokine
level is summarized as the log10 of the ratio relative to baseline (0 dpi), arctangent transformed to
reduce influence of extreme values. Rows (cytokines) are clustered using Ward's method and
squared Euclidean distance. Columns are grouped by time point sampled (d1, d3, d7), and ordered
by animal within time point, as labeled below the heatmap. Animal gender is indicated by text
color (blue, male; red, female). (b) Cytokines that exhibited significant changes from baseline are
indicated. Experiments were performed in duplicate (error bars represent SEM). The comparison
of the values from baseline was determined using a Kruskal-Wallis test with multiple
comparisons.
Supplementary Figure 8. ZIKV dynamics modeling. (a) Representative model fits (solid line)
to observed viral load in the plasma (filled circles) using a standard viral dynamic model with an
eclipse phase. The parameters fit were the rate of infection (β), rate of clearance of infected cells
(δ) and rate of production of virus from productively infected cells (p). Initial conditions, time
spent in the eclipse phase (1/k) and clearance of free virus (c) were fixed. (b) Correlation between
the observed plasma VL (log10) downslope (measured from peak observation to final detectable
observation) and CD16- CD56+ CD69+ NK cells (i) concentration at time of peak VL and (ii)
peak concentration but not (iii) concentration at time of infection. R and p values shown are from
the Pearson correlation, and p values are shown without correction for multiple testing. (c) The
half-life (mean 3.1 h) of a productively infected cell estimated from model fits to each monkey
(indicated by color) as in (b).
Nature Medicine: doi:10.1038/nm.4206
6
Supplementary Figure 9. ZIKV infectivity from the bodily fluids of infected macaques. Vero
cells were infected (at Day 0) using diluted semen, saliva or CSF. ZIKV RNA in culture
supernatants was assessed at various time points after infection. ZIKV-infected blood plasma
served as a positive control, mock infection as a negative control.
Supplementary Figure 10. Detection of ZIKV RNA in lymph node myeloid cells but not in T
or B cells. Axillary lymph nodes were stained for ZIKV RNA (red) by RNAscope and then
immunofluorescently stained for (left) CD163 and CD68 (green), DC-SIGN (blue), and DAPI on
day 5; (middle) CD163 and CD68 (green), CD20 (blue), and DAPI on day 7; and (right) CD163
and CD68 (green), CD3 (blue), and DAPI on day 5. Left image shows ZIKV RNA in
CD163/CD68+ (myeloid) cells, some of which also express DC-SIGN. Middle image shows
absence of ZIKV RNA in CD20+ (B) cells. Right image shows ZIKV RNA in CD163/CD68+
(myeloid) cells and absence of ZIKV RNA in CD3+ (T) cells. Images are representative of (n=2)
per time point. All images are shown at 60X magnification. Scale bar =200um.
Nature Medicine: doi:10.1038/nm.4206
a b c d
Supplementary figure 1
0 2 4 6 7 81 3 5 14 21102
3
4
5
6
7
8
Days post infection
Lo
g Z
IKV
RN
A c
op
ies/
ml
FemaleMale
0 7 14 213 102345678
Days post infection
Lo
g Z
IKV
RN
A
cop
ies/
ml
0 7 14 213 102345678
Days post infection
Lo
g Z
IKV
RN
A
cop
ies/
ml
0 7 14 212345678
Days post infection
Lo
g Z
IKV
RN
A
cop
ies/
ml
Nature Medicine: doi:10.1038/nm.4206
1.01.21.41.61.82.0
0 3 5 7
Alb
umin
/gl
obul
in (A
/G)
0
10
20
30
0 3 5 7
BU
N/
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atin
ine
(B/C
)
0.0
0.1
0.2
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Indi
rect
bili
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n(m
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L)
-0.050.000.050.100.15
0 3 5 7
Dire
ct b
iliru
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(mg/
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0.1
0.2
0.3
0.4
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Tota
l bili
rubi
n(m
g/d
L)
3
4
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Alb
umin
(g/d
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0
100
200
300
0 3 5 7
Alk
alin
eP
hosp
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se (U
/L)
15
20
25
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0 3 5 7
Bicarbonate
(mmol/L)
0
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20
30
0 3 5 7
Blo
od u
rea
nitr
ogen
(mg/
dL)
9
10
11
0 3 5 7
Cal
cium
(mg/
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100
105
110
115
0 3 5 7Chl
orid
e (m
mol
/L)
50
100
150
200
0 3 5 7Cho
lest
erol
(mg/
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0
2000
4000
6000
8000
0 3 5 7
Cre
atin
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osph
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ase
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)
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atin
ine
(mg/
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2.0
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3.5
0 3 5 7
Glo
bulin
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0
50
100
150
0 3 5 7
Glu
cose
(mg/
dL)
30
35
40
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0 3 5 7
NA/K
0
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4
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8
0 3 5 7
Phosphorous
(mg/dL)
3.0
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0 3 5 7Pot
assi
um (m
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0 3 5 7
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tein
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Supplementary figure 2
0
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0 3 5 7
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rase
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Nature Medicine: doi:10.1038/nm.4206
0 1 2 3 4 5 6 7 8 9 10 11 12 13 1434
35
36
37
38
39
40
Day 0 meantrough
Male meanFemale mean
Days post infection
Deg
rees
cel
sius
Supplementary figure 3
Nature Medicine: doi:10.1038/nm.4206
0.0
0.2
0.4
0.6
0.8
0 1 2 3 4 5 7
Eosinophils
(x10
3 /µL)
0.0
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n co
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Primary Infection
Re-infection
Supplementary figure 4
Nature Medicine: doi:10.1038/nm.4206
a b
Memory
01234
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01234
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# p
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NK Cells e
B Cells f
CD8+ T Cells d
CD4+ T Cells c
Days post infection
Days post infection
MedianFemaleMale
Days post infection
Monocytes
0 1 2 3 4 5 7 10 140
20
40
60
% C
D16
+
0 1 2 3 4 5 7 10 140
5
10
15
# p
er µ
L (x
102 )
Day 2
Males Females0
20
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% C
D16
+
P=0.0079
Supplementary figure 5
Nature Medicine: doi:10.1038/nm.4206
Capsid
0 7 14 280.0
0.1
0.2
0.3
% C
D10
7a
Capsid
0 7 14 280.0
0.5
1.0
1.5Envelope
0 7 14 28
Envelope
0 7 14 28
Capsid
0 7 1421280.0
0.1
0.2
0.3
% C
D10
7a
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0 7 1421280.0
0.5
1.0
1.5Envelope
0 7 142128
Envelope
0 7 142128
0 7 14 28
0 7 14 28
0 7 14 28
0 7 14 280.0
0.5
1.0
1.5
0 7 14 280.0
0.5
1.0
1.5 0 7 14 280.0
0.5
1.0
1.50 7 14 28
0 7 14 28
0 7 14 28
0 7 14 280.0
0.1
0.2
0.3
% IF
Nγ
0 7 14 280.0
0.1
0.2
0.3
% IL
-2
0 7 14 280.0
0.1
0.2
0.3
% T
NFα
0 7 142128
0 7 142128
0 7 142128
0 7 1421280.0
0.5
1.0
1.5
0 7 1421280.0
0.5
1.0
1.5 0 7 1421280.0
0.5
1.0
1.5
0 7 1421280 7 1421280.0
0.1
0.2
0.3
% IL
-2
0 7 142128
0 7 142128
0 7 1421280.0
0.1
0.2
0.3
% IF
Nγ
0 7 1421280.0
0.1
0.2
0.3
% T
NFα
Days post infection
CD4+ T Cells CD8+ T Cells
Days post infection
CD4+ T Cells CD8+ T Cells
a!
b!
Capsid
0 7 14 280.0
0.1
0.2
0.3
5169 (M)5690 (M)5818 (F)5829 (F)5939 (M)5963 (F)
5305 (M)5687 (M)5825 (F)5831 (F)
% C
D10
7a
Capsid
0 7 14 280.0
0.1
0.2
0.3
5169 (M)5690 (M)5818 (F)5829 (F)5939 (M)5963 (F)
5305 (M)5687 (M)5825 (F)5831 (F)
% C
D10
7a
Supplementary figure 6
Nature Medicine: doi:10.1038/nm.4206
MCP-1 IL-2 Il-15 VEGF IL-10 IL-1α IL-12/23(p40) IL-17α IL-1β MIP-1β G-CSF IL-5 GM-CSF IL-13 MIP-1α IL-6 TGF-α IL-8 sCD40L
-1 -0.5 0 0.5 1 atan Log10 fold change
200 100
0
a b
5829
d1
5690
d1
5305
d1
5963
d1
5939
d1
5687
d1
5818
d1
5169
d1
5831
d1
5825
d1
5829
d3
5690
d3
5305
d3
5963
d3
5939
d3
5687
d3
5818
d3
5169
d3
5831
d3
5825
d3
5829
d7
5690
d7
5305
d7
5963
d7
5939
d7
5687
d7
5818
d7
5169
d7
5831
d7
5825
d7
sCD40LIL8TGFaIL6MIP1aIL13GMCSFIL5GCSFMIP1bIL1bIL17aIL12/23(p40)IL1aIL10VEGFIL15IL2MCP1
−1 −0.5 0 0.5 1atan log10−fold change
010
020
0
Day 1 Day 3 Day 7
Supplementary figure 7
1 3 70
0.5
1
* **
IL-15
1 3 70
0.5
1**
VEGF
1 3 70
0.5
1
*
IL-10
1 3 70
1
2
3
****IL-1α
1 3 7-1
0
1
*
sCD40L
1 3 7-1
0
1
* *
IL-81 3 7
0
0.5
1
****
MCP-1
Log 1
0 Fol
d C
hang
es
Days post infection
Nature Medicine: doi:10.1038/nm.4206
Supplementary figure 8
a b
c
Nature Medicine: doi:10.1038/nm.4206
Supplementary figure 9
Log
ZIK
V R
NA
cop
ies/
ml
Hours post infection
0 24 48 722
3
4
5
6
7 Saliva
0 24 48 722
3
4
5
6
7 CSF
0 24 48 722
3
4
5
6
7 Blood
1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 20.0
5.0×107
1.0×108
1.5×108
Data 1
ControlSemen 1Semen 2Saliva 1Saliva 2CSF 1CSF 2Plasma
Plasma (Day 3)Mock
0 24 48 722
3
4
5
6
7 Semen
Nature Medicine: doi:10.1038/nm.4206
vRNA / CD163/CD68 / CD20 / DAPI vRNA / CD163/CD68 / DC-SIGN / DAPI vRNA / CD163/CD68 / CD3 / DAPI
Lymph Node
Supplementary figure 10
Nature Medicine: doi:10.1038/nm.4206
