Journal of General Virology (2015), 96, 3035–3048
DOI 10.1099/jgv.0.000246
Characterization of lethal dengue virus type 4
(DENV-4) TVP-376 infection in mice lacking
both IFN-a/b and IFN-c receptors (AG129)
and comparison with the DENV-2 AG129
mouse model
Vanessa V. Sarathy,1,2 Ernesto Infante,3 Li Li,1,2 Gerald A. Campbell,2
Tian Wang,1,2,4 Slobodan Paessler,1,2,5,6,7 P. Robert Beatty,8 Eva Harris,9
Gregg N. Milligan,1,3,4 Nigel Bourne1,3,4 and Alan D. T. Barrett1,2,5,6,7
Correspondence
1
Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, TX 77555,
USA
2
Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USA
3
Department of Pediatrics, University of Texas Medical Branch, Galveston, TX 77555, USA
4
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston,
TX 77555, USA
5
Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston,
TX 77555, USA
6
Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555, USA
7
Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch,
Galveston, TX 77555, USA
8
Division of Infectious Diseases and Vaccinology, School of Public Health, University of California
Berkeley, Berkeley, California, USA
9
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California,
USA
Alan D.T. Barrett
[email protected]
Received 1 May 2015
Accepted 6 July 2015
000246 G 2015 The Authors
Dengue is a mosquito-borne disease caused by four related but distinct dengue viruses,
DENV-1 to DENV-4. Dengue is endemic in most tropical countries, and over a third of the
world’s population is at risk of being infected. Although the global burden is high, no vaccine or
antiviral is licensed to combat this disease. An obstacle complicating dengue research is the
lack of animal challenge models that mimic human disease. Advances in immunocompromised
murine infection models resulted in development of lethal DENV-2, DENV-3 and DENV-4
models in AG129 mice, which are deficient in both the IFN-a/b receptor (IFN-a/bR) and the
IFN-c receptor (IFN-cR). These models mimic features of dengue disease in humans. Here, we
characterized lethal infection of AG129 mice by DENV-4 strain TVP-376 and found that AG129
mice developed clinical signs of illness and high viral loads in multiple tissues and succumbed
5 days after infection. Moreover, the splenic and hepatic histopathology of TVP-376-infected
mice demonstrated the presence of cell activation and destruction of tissue architecture.
Furthermore, infected mice had heightened levels of circulating cytokines. Comparison of the
virulence phenotypes of DENV-4 strain TVP-376 and DENV-2 strain D2S10 revealed that
TVP-376-induced mortality occurred in the absence of both IFN-a/bR and IFN-cR signalling,
but not with intact signalling from the IFN-cR, whereas D2S10 required the absence of
IFN-a/bR signalling only, indicating that it is more virulent than TVP-376. In conclusion,
TVP-376 is lethal in AG129 mice, and this model provides a useful platform to investigate
vaccine candidates and antivirals against DENV-4.
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3035
V. V. Sarathy and others
INTRODUCTION
The family Flaviviridae contains medically important
mosquito-borne viruses, including West Nile, yellow fever,
Japanese encephalitis and dengue virus types 1–4 (DENV-1
to -4). Dengue is endemic in most tropical countries, causing
an estimated 400 million infections annually (Messina et al.,
2014; Bhatt et al., 2013). Dengue fever is an acute illness with
symptoms that include myalgia, headache, retro-orbital
pain, maculopapular rash, vomiting and abdominal pain.
Severe forms of the disease are characterized by thrombocytopenia and vascular leakage, leading to dengue haemorrhagic fever (DHF). Dengue shock syndrome (DSS) is
characterized by hypovolaemic shock (Simmons et al.,
2012). Although DHF/DSS can be fatal, no treatments are
licensed to prevent or combat infections.
A limitation complicating dengue research is the lack of
animal challenge models that mimic human disease.
Recently, DENV-2 strains have been identified that produce
a disease that contains features of the human illness, such as
thrombocytopenia, viraemia, cytokine storm, and/or vascular leakage (Shresta et al., 2006; Tan et al., 2010; Mota & RicoHesse, 2009). Lethal DENV-2 infection models have been
described in immunocompromised mouse strains, particularly in AG129 mice, deficient in IFN-a/b receptor (IFN-a/
bR) and IFN-c receptor (IFN-cR) signalling, and A129 and
ifnar 2/2 mice, deficient in IFN-a/bR only (Johnson &
Roehrig, 1999; Orozco et al., 2012; Prestwood et al., 2012;
Zellweger et al., 2010; Balsitis et al., 2010). Other strains of
immunocompromised mice are resistant to lethal infection
by DENV-2, such as mice deficient in IFN regulatory factors
3 and 7 (IRF3 and IRF7), signal transducer and activator of
transcription factor 1 or 2 (STAT1 or STAT2), and mitochondrial antiviral signalling protein (MAVS) (Chen et al.,
2013; Perry et al., 2009, 2011; Ashour et al., 2010).
Lethal models for DENV-3 and DENV-4 in AG129 mice have
also been reported. A non-adapted human isolate of DENV3 (strain C0360/94) was found to cause severe systemic nonneurotropic infection, characterized by thrombocytopenia,
vascular leakage, circulating cytokines, and TNF-a-mediated
disease (Sarathy et al., 2015). Mouse-adapted DENV-4
strains derived from H241 and TVP-376 were used to
study antibody protection and neutralization (SukupolviPetty et al., 2013). These adapted viruses led to neurovirulent
disease and for TVP-376 mortality increased with each of
two passages, indicating that a single mouse passage can
influence DENV-4 disease in AG129 mice. Finally, a new
study described the virulence of non-adapted DENV-4
strain 703-4 in AG129 mice (Milligan et al., 2015). The animals succumbed to a lethal, non-neurovirulent systemic disease and developed elevated cytokine levels and vascular
leakage. Recently, this 703-4 model was used to evaluate a
candidate live attenuated dengue vaccine. The tetravalent
dengue vaccine reduced viraemia and protected mice against
lethal 703-4 challenge (Fuchs et al., 2014).
Subsequently, we determined that non-mouse-adapted
DENV-4 strain TVP-376 causes systemic disease in AG129
3036
mice similar to that caused by DENV-4 strain 703-4. In the
current study, we present a characterization of this lethal
TVP-376 infection in AG129 mice, and the sensitivity of
WT, Mavs 2/2, A129 and AGB6 mice to non-adapted TVP376 is evaluated. Although DENV-2 has been examined
previously in these mouse strains, there is variation in the
viral strains and viral quantification methods (infectivity
versus genome copies). Therefore, a side-by-side comparison
between DENV-4 TVP-376 and DENV-2 D2S10 is
included. The objectives of this study were to evaluate lethal
TVP-376 infection and to provide a platform to compare
the mechanisms of DENV-2 and DENV-4 infection in
AG129 mice.
RESULTS
Derivation of DENV-4 strain TVP-376
The DENV-4 reference strain TVP-376 was acquired from
Dr Morag Ferguson of the National Institute of Biological
Standards and Control (NIBSC), Potters Bar, UK, who
received the virus from Dr Robert Putnak of the Walter
Reed Army Institute for Research. This strain was isolated
from a clinical acute serum sample of infection acquired in
Puerto Rico in 1982 (R. Tesh, personal communication).
Unfortunately, confusingly, TVP-376 has been mislabelled
as TVP-360 and has also been mistakenly described as a
Colombian isolate (Sukupolvi-Petty et al., 2013).
DENV-4 TVP-376 is lethal in AG129 mice
DENV-4 TVP-376 was tested in AG129 mice (6–8 weeks old)
at doses comparable with those used for the other AG129
DENV models (Shresta et al., 2006; Sarathy et al., 2015; Milligan et al., 2015). With an inoculum of 107.0 p.f.u. or higher,
100 % of the mice succumbed within 1 week of infection
(Fig. 1a). Animals lost weight starting at 3 days post-infection
(days p.i.) (Fig. 1b) and exhibited physical signs of illness, such
as limited mobility and hunched posture (data not shown).
Only one case of neurological disease occurred – one mouse
developed paralysis at 7 days p.i. Median survival times for
107.5 p.f.u.-infected and 107.0 p.f.u.-infected mice were 3.5
and 5.0 days, respectively. All AG129 mice infected with
106.0 p.f.u. TVP-376 survived, and did not lose weight or
have detectable viraemia (Fig. 1b, c). An equivalent dose–
response using DENV-2 strain D2S10 resulted in similar outcomes. Inoculation of 106.0, 107.0 and 107.5 p.f.u. D2S10
resulted in 14, 87 and 100 % mortality, respectively
(Fig. 1a). Together, these results show that TVP-376 leads to
lethal disease in AG129 mice with mortality similar to
D2S10, and that both viruses have an LD50 of 106.5 p.f.u.
Lethal TVP-376 infection of AG129 mice is
characterized by tissue viral loads
To characterize lethal infection by 107.0 p.f.u. DENV-4
strain TVP-376, groups of 3–5 animals were sacrificed on
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Journal of General Virology 96
Lethal DENV-4 model in AG129 mice using strain TVP-376
(a)
(b)
110
Body weight (%)
100
Survival (%)
75
50
100
90
80
25
P<0.0001
70
0
0
5
10
15
Days p.i.
D2S10 106.0 p.f.u (n=6)
D2S10 10
7.0
p.f.u (n=7)
D2S10 10
7.5
p.f.u (n=8)
20
25
30
0
TVP-376 106.0 p.f.u (n=8)
TVP-376 10
7.0
p.f.u (n=10)
TVP-376 10
7.5
p.f.u (n=2)
(c)
4
6
Days p.i.
TVP-376 106.0 p.f.u (n=8)
8
10
12
TVP-376 107.0 p.f.u (n=10)
7.5
TVP-376 10
TVP-376
106.0 p.f.u.
5
2
p.f.u (n=2)
TVP-376
107.0 p.f.u.
log10(p.f.u. ml–1)
4
3
2
1
0
1
2
2
(n=4)
(n=4)
(n=3)
Days p.i.
Fig. 1. DENV-4 TVP-376 is lethal in AG129 mice. (a) Kaplan–Meier survival curves of AG129-mice infected with 106.0,
107.0 or 107.5 p.f.u. D2S10 or TVP-376. #, Mouse developed paralysis. (b) Mean percentage weight lost¡SE relative to animal weight of TVP-376-infected mice (107.0 p.f.u.: ANOVA P,0.0001; Dunnett’s post-test, significant at 3–6 days p.i.). (c)
Viraemia titres (mean¡SE ) of mice infected with 106.0 and 107.0 p.f.u. TVP-376.
each day (1, 2, 3 and 4 days p.i.), and viral loads were
assessed in serum, liver, spleen, large intestine and brain.
At 1 day p.i., 71 % of the animals had infectious virus in
the serum and visceral organs; this rose to 100 % of animals at 2, 3 and 4 days p.i. (Fig. 2a). The liver, spleen
and large intestine contained high infectivity titres 2–
4 days p.i. (mean 105–106.5 p.f.u. g21). In contrast, virus
was detected in the brain of only 14 % of animals (1/7)
at 1 and 2 days p.i., increasing to 71 % (5/7) on day 3,
and falling to 40 % (2/5) on day 4. Brain infectivity was
consistently lower than in other tissues.
TVP-376 antigen was detected by fluorescence immunohistochemistry. Immunostaining of liver and spleen sections
with anti-NS1 indicated that TVP-376 was present and
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replicating in infected tissues throughout the course of
infection (days 1 and 3 shown; Fig. 2b, c). In the spleen,
the viral antigen appeared in cells at the margins of the follicles at 1 day.p.i., and the immunoreactive cells were diffusely distributed by 3 days p.i. (i.e. they were not localized to
a structurally delineated area of the spleen). No virus antigen was detected in mock-infected controls. Together,
these data indicate that TVP-376-infected AG129 mice
develop systemic infection.
AG129 mice infected with a lethal dose of TVP-376
develop leukopenia but not thrombocytopenia
Blood samples from mock-infected (n510) or TVP-376infected AG129 mice (107.0 p.f.u.) were collected at 1 (n53)
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V. V. Sarathy and others
(a)
log10(p.f.u. ml–1 or g–1)
Serum
Liver
Spleen
*
*
7
6
5
4
3
2
1
0
1
2
3
4
1
2
3
4
1
Large intestine
Brain
*
*
2 3 4
Days p.i.
1
2
3
4
1
2
3
4
(b)
1 days p.i.
3 days p.i.
Spleen (×10)
Liver (×20)
Mock
(c)
Liver 1 day p.i. (×100)
Isotype
Anti-NS1
Liver 1 day p.i. (×60)
Fig. 2. Lethal TVP-376 infection in AG129 mice is characterized by high viral loads in tissues. (a) Infectivity titres of serum
and tissue samples from AG129 mice infected with 107.0 p.f.u. Data represent individual titres (symbols), mean daily titre
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Journal of General Virology 96
Lethal DENV-4 model in AG129 mice using strain TVP-376
(lines), and statistical significance (asterisks) analysed using ANOVA with Tukey’s post-test. Results are combined from two
separate studies: serum, liver, spleen and brain, n57 for days 1–3 and n55 for day 4; large intestine, n53 for days 1–3, and
n55 for day 4. (b) Detection of DENV NS1 protein in liver (upper panels) and spleen (lower panels) sections of AG129 mice
infected with 107.0 p.f.u. TVP-376; nuclei, blue; NS1, green; liver and spleen sections shown at 620 and 610 magnification,
respectively. (c) Liver sections from 1 day p.i. immunostained with anti-NS1 (top panels) or isotype (bottom panels) at 660
and 6100.
and 3 days p.i. (n53). Compared with controls, TVP376 infection resulted in leukopenia as early as 1 day p.i.
(Pv0.01), including decreased lymphocyte counts
(Pv0.001), which continued throughout infection (Fig. 3).
Significantly, no changes were observed in platelet numbers
or in the haematocrit percentage. These results indicate
that lethal TVP-376 infection of AG129 mice includes some
signs of human disease but no significant thrombocytopenia.
Lethal TVP-376 infection causes histopathology in
the spleen and liver of AG129 mice
Histological examination was undertaken to further investigate the course of infection. Tissues were harvested after
TVP-376 infection (at 1–4 days p.i.), and histopathological
changes were observed in the liver and spleen (Fig. 4a–i),
but not in large intestine or brain tissues (data not
shown). Liver haematoxylin and eosin (H&E) sections
were graded, and the plotted scores indicate the presence
of tissue damage and cellular activation (Fig. 4l). Representative panels show increased focal necrosis, decreased glycogen content, and significant nuclear pleomorphism or
binucleation of hepatocytes (Fig. 4c–e) (Pv0.05). Portal
venule thrombosis was also observed (Fig. 4c, d).
Additional liver sections were stained with periodic acid–
Schiff stain (PAS) to confirm that glycogen stores were
depleted in TVP-376-infected hepatocytes (Fig. 4j, k)
(Pv0.0001) (scores not shown). Also, kinetic histological
changes were detected in spleen H&E sections. Initially
(1–2 days p.i.), the haematopoietic cell content and red
pulp areas increased (Pv0.01), but they declined at 3
and 4 days p.i. (Fig. 4g–i, m). This change was countered
with an increase in the white pulp and germinal centre
cell transformation of the white pulp; both showed a
steady, time-dependent increase and peaked at 4 days p.i.
(Pv0.05 and Pv0.0001, respectively). Furthermore, infection led to a statistically significant (Pv0.001) increase in
spleen weight by 2 days p.i., from 0.28 to 0.45 % of body
Platelets
800
Haematocrit
60
400
%
× 103 cells ml–1
80
600
40
200
20
0
0
Control
1
3
Control
Leukocytes
3
Lymphocytes
15
15
**
× 103 cells ml–1
*
× 103 cells ml–1
1
10
5
0
**
***
1
3
10
5
0
Control
1
3
Control
Fig. 3. TVP-376-infected AG129 mice exhibit leukopenia but not thrombocytopenia. Blood counts of naive (n510) or
107.0 p.f.u.-TVP-376-infected samples harvested at 1 and 3 days p.i. (n53 per group); platelet, leukocyte, and lymphocyte
numbers and haematocrit percentage. Data represent individual values (symbols), mean¡SE (lines), and statistical significance (asterisks) analysed using ANOVA with Dunnett’s post-test.
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3039
V. V. Sarathy and others
(a)
Liver H&E stain
Spleen H&E stain
(f)
Liver glycogen stain
(j)
WP
(c)
d
RP
e
(b)
(g)
(d)
(h)
(k)
(l)
M
Score
NP
Liver histopathology
2
1
0
–1
–2
G
NP/BN
FN
Spleen histopathology
(m)
PVT
Score
2
(e)
0
–1
(i)
FN
1
0
1
2
3
4
Days p.i.
RP
H
GC
GC
Splenomegaly
% Body weight
(n)
NP
W
*** **** ***
0.6
0.4
0.2
0
0
1 2 3
Days p.i.
4
Fig. 4. Histology of 107.0 p.f.u.-TVP-376-infected AG129 mice shows damage to liver and splenomegaly due to lymphoid
hyperplasia. H&E stains of tissue sections harvested at 1–4 days p.i. from TVP-376-infected AG129 mice (n526) were compared and scored relative to those from naive and mock-infected mice (n58). Panels are representatives from two
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Journal of General Virology 96
Lethal DENV-4 model in AG129 mice using strain TVP-376
independent studies. Liver sections from mock-infected (a, b) and TVP-376-infected 1 day p.i. samples (c–e) were evaluated
for glycogen content (G), nuclear pleomorphism (NP), and binucleation (BN), focal necrosis (FN) and portal venule thrombosis (PVT). Magnification: 64 (a, c); 610 (b, d, e). Spleen section H&E stains from mock-infected (f), 1 (g), 2 (h) or 3 days
p.i. (i) Samples show white pulp (WP) and red pulp (RP) content of the spleen and megakaryocytes (M), indicators of haematopoietic cell (H) activity and germinal center cell transformation (GC). PAS-stained liver sections from a mock-infected (j)
and TVP-376-infected (k) sample show depletion of glycogen content (absence of intense pink staining). (l, m) Graphical representations of semiquantitative scores of H&E-stained sections from a total of 26 infected mice. (l) Box and whisker plot of
semiquantitative scores of liver sections. (m) Mean scores¡SE of spleen sections. Significance was determined using
ANOVA and Dunnett’s post-test to compare scores from day 0 with days 1–4 and are denoted in green, red, blue and purple
asterisks for H, RP, GC and WP, respectively. Spleen weights at necropsy were plotted, and horizontal lines represent the
mean percentage of total body weight; significance analysed using ANOVA and Dunnett’s post-test. Scale bars, 50 mm.
weight, peaking at 3 days p.i. at 0.55 % (Fig. 4n), resulting
in splenomegaly, which may be caused by the proliferative
activation and cellular expansion occurring in the organ
during TVP-376 infection.
the median survival time from 5 to 13 days, it was not statistically significant (P50.3383) (Fig. 5c). Furthermore,
anti-TNF-a-treated animals that succumbed to infection
after 10 days p.i. (n53) developed paralysis.
AG129 mice infected with TVP-376 develop
increasing circulating cytokine levels until death
Mavs 2/2 mice are resistant to both D2S10 and
TVP-376 infection
In order to evaluate the innate immune response to TVP376 infection, sera from AG129 mice infected with
107.0 p.f.u. were analysed by Bioplex analysis (23-plex)
(Fig. 5a). Compared with mock-infected controls, levels
of 15 cytokines were significantly increased, while levels
of six were comparable to controls (IL-1b, IL-2, IL-3,
IL-4, IL-5 and IL-12p70; data not shown); Eotaxin and
granulocyte–monocyte-colony-stimulating factor were excluded from the analysis because their values were outside the
detectable range for the majority of the mock- and virusinfected animals. Chemokine (C-X-C motif) ligand 1
(CXCL1), chemokine (c-c motif) ligand 2 (CCL2), CCL3,
CCL4 and CCL5 were all elevated during infection. Inflammatory cytokines IL-1a, IL-6, IL-12p40, IL-17A, IFN-c and TNF-a
were elevated, although the latter only showed significant
elevation at 3 days p.i. Changes in serum levels of cytokines
involved in cell growth, proliferation, and differentiation
were also detected (IL-9 and granulocyte-colony-stimulating
factor). Anti-inflammatory cytokines were also elevated:
IL-10 on all days, and IL-13 later during infection. Thus, the
lethal TVP-376 AG129 model leads to increased levels of cytokines that have been identified as markers for human dengue
infection, including IL-6, CXCL1 and IFN-c (Srikiatkhachorn
& Green, 2010; Bozza et al., 2008; Rothman, 2011). The kinetics
of the cytokine response to TVP-376 showed that at 1, 2 and
3 days p.i. the number of elevated cytokines increased from 5
to 10 to 15, respectively, indicating that the mechanism of
TVP-376 infection involves overwhelming cytokine activation
and release (Fig. 5b).
In order to determine if DENV-4 strain TVP-376 uses a
similar mechanism to DENV-2 for mouse mortality, several comparisons were performed. The role of MAVS in
DENV-2 infection has been examined using 1012 genomic
equivalents of S221, a strain related to D2S10, but not in
DENV-4 infection (Perry et al., 2009). Therefore,
107.5 p.f.u. D2S10 or TVP-376 was inoculated into 8week-old WT and Mavs 2/2 mice. Neither virus induced
mortality or weight loss in WT or Mavs 2/2 mice, and
examination of viraemia indicated that WT mice were
not infected at 3 days p.i. (Fig. 6a–c). A single D2S10infected Mavs 2/2 mouse had detectable viraemia; whereas
the majority of TVP-376-infected Mavs 2/2 mice had viraemia of approximately 102 p.f.u. ml21. Lastly, neutralization
assays of sera harvested at 30 days p.i. indicate that both
WT and Mavs 2/2 mice generate significantly higher neutralizing antibody titres to D2S10 [1272, 95 % confidence
interval (CI) 1021–1585; 1255, 95 % CI 1154–1366,
respectively] than to TVP-376 (321, 95 % CI 247–417;
898, 95 % CI 760–1063, respectively) (Pv0.0001). These
results show that loss of MAVS signalling is not a major
contributor to murine lethality by either D2S10 or TVP376 because the animals showed no weight loss, had low
viraemia at 3 days p.i. and survived infection.
TNF-a contributes to lethal infection in humans and in
AG129 mouse models of DENV-2 (Srikiatkhachorn &
Green, 2010; Atrasheuskaya et al., 2003; Shresta et al.,
2006; Sarathy et al., 2015). Administration of anti-TNF-a
antibody or isotype control (groups of five mice) into
AG129 mice infected with TVP-376 resulted in 20 and
0 % survival, respectively; although the treatment increased
The lack of signalling from the IFN-a/bR and IFN-cR renders AG129 mice susceptible to lethal infection by DENV,
including TVP-376 (Fig. 1a). Because both the 129 and B6
mouse backgrounds are frequently used in DENV-2 susceptibility studies, a comparison of AGB6 mice infected
with D2S10 or TVP-376 was performed. AGB6 mice were
highly susceptible to 107.4 p.f.u. of each virus, with a
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Mice deficient in IFN receptors have higher
susceptibility to D2S10 than to TVP-376
infection
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3041
V. V. Sarathy and others
pg ml–1
(a)
IL-6
IL-1a
50
40
30
20
10
0
250
200
150
100
50
0
**
**
0
1
2
3
*
pg ml–1
pg ml–1
pg ml–1
2
0
3
IL-17A
***
**
10
104
103
102
101
100
**
50
500
400
300
200
100
0
0
1
2
3
CXCL1
*
5000
4000
3000
2000
1000
0
**
**
0
1
2
3
0
pg ml–1
100
0
50
*
*
*
1
1
*
2
0
3
2
***
**
300
250
200
150
40
20
3
**
*
**
0
3
**
2
3
1
0
2
3
1
2
Days p.i.
*
100
80
60
40
20
0
*
*
*
*
0
1
2
3
TNF-a
*
300
*
**
**
200
100
**
0
1
CCL3
CCL5
***
*
200
0
IFN-g
**
0
0
3
400
**
1
2
Days p.i.
2
CCL2
*
600
**
1
G-CSF
**
*
**
800
0
IL-13
100
CCL4
150
0
3
200
5
100
0
2
**
1
200
1
300
200
**
150
0
**
*
0
0
3
400
20
0
2
600
**
**
40
100
IL-12p40
**
60
1
*
200
*
0
*
300
*
*
IL-10
80
IL-9
0
3
0
1
2
Days p.i.
Naive
Mock
3
TVP-376
(c)
(b)
20
Survival (%)
No. cytokines
25
15
10
5
0
1
2
Days p.i.
Increased
expression
3
No change
100
80
60
40
20
0
#
P=0.34
#
#
10
20
Days p.i.
0
30
Anti-TNF-a (n=5)
Isotype control (n=5)
Not analysed
3042
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Journal of General Virology 96
Lethal DENV-4 model in AG129 mice using strain TVP-376
Fig. 5. TVP-376 infection in AG129 mice leads to kinetic cytokine responses but not to TNF-a-mediated lethality. (a) Bioplex
was performed on sera harvested from naive (n58), mock-infected 1–3 days p.i. (n55), and 107 p.f.u. TVP-376-infected
1–3 days p.i. (n57) AG129 mice from two separate experiments. Bars represent means¡SE . Cytokine levels from virusinfected animals were compared with same-day-matched mock-infected controls using the Mann–Whitney U-test (asterisks
above individual bars). Values of infected samples for 1–3 days p.i. were compared using Kruskal–Wallis ANOVA (dashed
line spanning the top of the graph) and multiple comparisons (solid lines connecting two bars). (b) Bioplex results were
sorted by number of cytokines that were significantly elevated, unchanged, or not analysed, then plotted by day p.i.
(c) Kaplan–Meier survival curves of AG129 mice infected with 107 p.f.u. TVP-376 and then administered either functional
grade anti-TNF-a (n55) or isotype control (n55) for 3 days. #, Mouse developed paralysis.
lethargy, while the 107.4 p.f.u.-D2S10-infected mice
demonstrated more severe signs of disease, including
hunched posture, diarrhoea and minimal mobility. The
AGB6 mice that survived TVP-376 infection showed clinical signs of illness at 4–9 days p.i. but recovered, and had a
mean terminal neutralizing antibody titre of 913 (95 % CI
727–1148; data not shown). All groups were viraemic at
median survival of 3.0 days (Fig. 7a). Infection with a lower
inoculum of 106.4 p.f.u. resulted in 100 and 50 % mortality
and median survival times of 4.0 and 18.5 days (P50.0082,
log-rank test) for D2S10 and TVP-376, respectively. All
AGB6 mouse groups had significant weight loss starting
at 3–4 days p.i. (Fig. 7b). In addition, the 107.4 p.f.u.TVP-376-infected mice exhibited hunched posture and
(a)
(b)
110
Body weight (%)
Survival (%)
100
75
50
25
100
90
80
70
0
0
5
10
15
20
Days p.i.
25
0
30
2
4
Days p.i.
6
8
D2S10 WT (n=3)
D2S10 Mavs–/– (n=4)
D2S10 WT (n=3)
D2S10 Mavs–/– (n=4)
TVP-376 WT (n=3)
TVP-376 Mavs–/– (n=4)
TVP-376 WT (n=3)
TVP-376 Mavs–/– (n=4)
(c)
log10(p.f.u. ml–1)
5
Mavs–/–
WT
4
3
2
1
6
P37
TV
10
D
2S
6
P37
TV
D
2S
10
0
Fig. 6. WT and Mavs 2/2 mice are resistant to D2S10 and TVP-376. (a) Kaplan–Meier survival curves of WT and Mavs 2/2
mice infected with 107.5 p.f.u. D2S10 or TVP-376. (b) Mean percentage weight lost¡SE relative to animal weight. (c) Viraemia
titres (mean¡SE ).
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3043
V. V. Sarathy and others
(a)
(b)
110
Body weight (%)
Survival (%)
100
75
50
100
90
80
25
70
0
0
5
10
6.4
D2S10 10
D2S10 10
15
20
Days p.i.
p.f.u. (n=4)
7.4
p.f.u. (n=5)
25
6.4
TVP-376 10
7.4
TVP-376 10
0
30
2
6.4
D2S10 10
p.f.u. (n=4)
D2S10 10
6
TVP-376 10
p.f.u. (n=5)
7.4
TVP-376 10
p.f.u. (n=4)
p.f.u. (n=5)
(d)
AGB6
107.4p.f.u.
AGB6
106.4p.f.u.
TVP-376 107.4 p.f.u.
***
5
*
8
log10(p.f.u. g–1)
4
3
2
1
4
2
n
ai
Br
e
In
te
st
in
en
r
ve
le
Sp
TV
P-
37
10
2S
D
TV
P-
37
10
2S
D
6
0
6
0
6
Li
log10(p.f.u. ml–1)
8
6.4
p.f.u. (n=4)
7.4
p.f.u. (n=5)
(c)
4
Days p.i.
Fig. 7. AGB6 mice are susceptible to D2S10 and TVP-376 infection. (a) Kaplan–Meier survival curves of AGB6 mice
infected with 106.4 or 107.4 p.f.u. D2S10 or TVP-376. (b) Mean percentage weight lost¡SE relative to animal weight.
ANOVA and Dunnett’s post-test: 106.4 p.f.u. D2S10, P,0.0001 (significant at 3 and 4 days p.i.); 106.4 p.f.u. TVP-376,
P,0.0001 (significant at 4–6 days p.i.); 107.4 p.f.u. TVP-376, P,0.01 (significant at 3 and 4 days p.i.); t-test of 107.4 p.f.u.
D2S10 between 0 and 3 days p.i., P,0.0001. (c) Viraemia titres of AGB6 mice assessed at 3 days p.i. (d) Infectivity titres of
tissues harvested from AGB6 mice infected with 107.4 p.f.u. TVP-376 at moribund state (3–4 days p.i.) (n54). Data represent individual titres (symbols), mean daily titre (lines), and statistical significance (asterisks) analysed using ANOVA with
Tukey’s post-test.
3 days p.i., and the mean titres of D2S10-infected mice
were slightly higher than those of TVP-376-infected:
106.4 p.f.u.-infected, 104.6 and 102.4 p.f.u. ml21, respectively; 107.4 p.f.u.-infected, 104.6 and 104.2 p.f.u. ml21,
respectively (Fig. 7c). Lastly, the systemic TVP-376 viral
burden of moribund AGB6 mice infected with 107.4 p.f.u.
was evaluated (Fig. 7d). The liver, spleen, intestine and
brain had viral loads of 106.0, 107.4, 106.4 and 104.8 p.f.u. g21,
respectively. The titres in the intestine and spleen were significantly higher (Pv0.001, ANOVA) than in the brain, and no
neurological clinical signs were observed. Taken together,
these results show that AGB6 mice are highly sensitive to infection by both D2S10 and TVP-376, and this direct comparison
3044
shows that D2S10 kills 100 % of mice at a lower inoculum
than TVP-376; therefore, it is more virulent than TVP-376,
with respective LD50 values of 105.9 and 106.6 p.f.u.
IFN-cR signalling is sufficient for mouse
resistance to TVP-376, but resistance to D2S10
requires both IFN-a/bR and IFN-cR
Studies with DENV-2 strains have shown that AG129 mice are
more susceptible to infection than A129 mice (Orozco et al.,
2012; Prestwood et al., 2012). However, no studies have
been conducted with the other DENV serotypes. A129 mice
were tested for susceptibility to 107.0 p.f.u. TVP-376,
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Journal of General Virology 96
Lethal DENV-4 model in AG129 mice using strain TVP-376
but none (n510) succumbed. In comparison, 75 % (n53) of
A129 mice infected with D2S10 (107.4 p.f.u.) died. Thus,
D2S10 but not TVP-376 is lethal in A129 mice (Fig. 8a)
(log-rank test P50.0020). Notwithstanding, TVP-376infected A129 mice lost significant weight by 1 day p.i.
(Pv0.0001), dropping to 87 % of original weight on day 4,
but regained weight to normal levels by 11 days p.i. (Fig.
8b). Also, A129 mice infected with D2S10 or TVP-376 had
indistinguishable viraemia on days 1 and 2 p.i. (Fig. 8c). Furthermore, 2 days p.i. viraemia of AG129 (103.4 p.f.u.) and
A129 (102.8 p.f.u.) infected with TVP-376 trended towards a
difference but was not statistically significant (t-test
P50.0794). Therefore, inoculation of D2S10 or TVP-376
into either A129 or AG129 mice results in virus infection,
but only D2S10 leads to mortality in A129 mice.
Recent studies with DENV-2 models have made major
contributions to this field (Mota & Rico-Hesse, 2009;
Tan et al., 2010; Shresta et al., 2006). However, all four
DENVs have been implicated in worldwide disease, and
limited information is available that distinguishes murine
models of different DENVs.
The lethal DENV-2 D2Y98P and D2S10, DENV-3 C0360/
94 and DENV-4 703-4 mouse models have been established
and characterized in AG129 mice; therefore, the AG129
model was chosen to further dissect the mechanism of
TVP-376 virulence. Similarly to the above-mentioned
DENV mouse models, TVP-376 infection leads to high
viral loads and replicates in several tissues (Fig. 3). However, unlike D2S10 and C0360/94, TVP-376 infection did
not cause thrombocytopenia (Fig. 3).
DISCUSSION
Histopathological changes occurred during TVP-376 infection. Liver sections contained hepatocytes with nuclear
pleomorphism and areas of necrosis, similar to the
D2Y98P, C0360/94 and 703-4 models (Tan et al., 2010; Sarathy et al., 2015; Milligan et al., 2015). Also, TVP-376
Anti-dengue therapies and vaccines are needed to address
the growing global health threat. One obstacle has been
the lack of suitable animal models to test DENV infection
challenge (Zompi & Harris, 2012; Yauch & Shresta, 2008).
(a)
(b)
110
Body weight (%)
Survival (%)
100
75
50
25
P<0.0001
100
90
*
80
*
*
*
* *
*
*
*
*
70
0
0
5
10
15
20
Days p.i.
D2S10 (n=4)
25
30
0
2
4
6
8
Days p.i.
10
12
TVP-376 (n=10)
TVP-376 (n=10)
log10(p.f.u. ml–1)
(c)
5
D2S10
TVP-376
4
**
*
3
2
1
0
1
(n=2)
2
(n=2)
1
(n=2)
2
(n=2)
Days p.i.
Fig. 8. TVP-376 infects A129 mice but is not lethal. (a) Kaplan–Meier survival curves of A129 mice infected with 107.4 p.f.u.
D2S10 or 107.0 p.f.u. TVP-376.(b) Mean percentage weight lost¡SE relative to animal weight (ANOVA P,0.0001; Dunnett’s
post-test, significant at 2–10 days p.i.). (c) Viraemia titres of mice infected with 107.4 p.f.u. D2S10 or 107.0 p.f.u. TVP-376;
statistical significance (asterisks) between 1 and 2 days p.i. analysed using t-test).
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3045
V. V. Sarathy and others
infection destroyed the splenic architecture and led to splenomegaly, highly proliferative cellular activation, and white
pulp expansion (Fig. 4). To this end, the presence of NS1 in
spleen sections at 1 day p.i. was restricted to the areas surrounding the follicles, but at 3 days p.i. the staining pattern
was distributed throughout the organ (Fig. 2b). This splenic phenotype is similar to but more severe than that
caused by C0360/94 and D2S10 infections, and resembles
DENV-2 D2Y98P and 703-4 infection (Sarathy et al.,
2015; Shresta et al., 2006; Tan et al., 2010; Milligan et al.,
2015).
Several cytokines previously identified as markers of
dengue infection in human patients and other models of
infection were significantly elevated in TVP-376-infected
AG129 sera, including IL-6, IL-8, IL-10, IFN-c and TNFa (Fig. 5a) (Srikiatkhachorn & Green, 2010; Bozza et al.,
2008; Rothman, 2011). Also, the number of elevated cytokines increased throughout the course of infection (Fig. 5b).
One difference among AG129 models is the kinetics of
TNF-a induction. D2Y98P, D2S10, 703-4 and TVP-376
cause a time-dependent increase of TNF-a, but C0360/94
induces high levels of TNF-a beginning at 1 day p.i.
(Tan et al., 2010; Shresta et al., 2006; Milligan et al., 2015;
Sarathy et al., 2015). Neutralization of TNF-a in TVP-376infected mice increased the median survival time from 5 to
13 days p.i. but the increase was not statistically significant
(Fig. 6c). Similarly, anti-TNF-a delayed D2S10 mortality
(from 4 to 10 days p.i., Pv0.0001) (Shresta et al., 2006),
whereas it completely rescued mice from lethal C0360/94
infection (Pv0.0001) (Sarathy et al., 2015). These differences
in survival and anti-TNF-a rescue could be attributed to the
notion that TNF-a plays a different role in the disease mechanism depending on whether it is elevated early (1 day p.i.) or
later (3–4 days p.i.) during the course of infection.
Mouse-passaged TVP-376 has been used at low doses of
104–105 p.f.u. to infect AG129 mice, but infections resulted
in an extended mean time to death and neurological disease (Sukupolvi-Petty et al., 2013). The present study represents, to the best of our knowledge, the first detailed
characterization of a non-mouse-adapted TVP-376 model
in AG129 mice, as the infecting virus was only amplified
three times in mosquito cells to obtain working stocks
and to avoid mammalian adaptation. Notably, unlike in
Sukupolvi-Petty et al. (2013), AG129 mice infected with a
lower dose (106.0 p.f.u.) did not develop neurological
disease up to 28 days p.i. Furthermore, none of the
lethal-dose-infected AG129 mice sampled at 1–4 days p.i.
exhibited any brain pathology. The few mice that had
detectable infectivity in the brain had lower titres than
detected in other organs, suggesting that virus detected in
the brain may be from the viraemia. In the present study,
lethal-dose-infected AG129 mice with delayed death or
prolonged survival developed paralysis (Figs 1a and 5c).
This phenomenon was restricted to AG129 mice; none of
the surviving AGB6 mice developed paralysis. These differences suggest that delayed infection with non-adapted
TVP-376 may ultimately lead to neurovirulence in
3046
AG129, an observation that has also been made in
D2S10-infected AG129 mice, but not in AGB6. These
results are similar to those observed previously for D2S10
but not for C0360/94.
Decades of experiments using immunocompetent mice to
establish DENV infection have shown that these mice are
generally resistant to lethal infection. This can be explained
at least in part by differences between the mouse and the
human immune response to DENV (Green et al., 2014;
Zellweger & Shresta, 2014; Ashour et al., 2010; Aguirre
et al., 2012). In the present study, WT mice inoculated
with D2S10 or TVP-376 survived infection, showed no
clinical signs of disease, and had no detectable viraemia.
MAVS has been shown to play a role in IFN induction in
the hours after infection with DENV-2 S221; however,
other factors control IFN signalling at later times, rendering Mavs 2/2 mice resistant to lethal infection (Perry
et al., 2009). In the present study, DENV-2 D2S10 and
DENV-4 TVP-376 were inoculated into Mavs 2/2 mice in
order to compare their virulence. All animals survived
infection, but more TVP-376-infected than D2S10-infected
mice were viraemic at 3 days p.i. (Fig. 6a–c). This suggests
that the kinetics of infection by the two viruses may differ
in Mavs 2/2 mice; however, detailed studies would be
required to determine whether this is a virus strain-specific
or a virus serotype-specific phenomenon.
Infection of C57BL/6 mice deficient in both IFN-a/bR and
IFN-cR (AGB6) by D2S10 and TVP-376 resulted in the
expected results of viral infectivity and death. Notably,
mice infected with D2S10 suffered more severe disease,
accompanied by faster and higher mortality, leading to a
slightly lower LD50 (105.9 p.f.u) compared with TVP-376
(106.6 p.f.u). Hence, D2S10 is fivefold more virulent than
TVP-376 in AGB6 mice, whereas the LD50 for D2S10 and
TVP-376 in AG129 mice was the same (106.5 p.f.u.). Therefore, these data suggest that there are genetic differences
between the two mouse strains that affect DENV infection.
Detailed studies showed that IFN-cR-mediated responses
protect mice from paralysis resulting from DENV-2 infection (Shresta et al., 2004; Prestwood et al., 2012) Previously, AG129-virulent DENV-3 and DENV-4 strains
had not been evaluated in the absence of the IFN-a/bR;
so in the current report, a comparison of D2S10 and
TVP-376 in A129 mice was performed. As expected,
D2S10 causes lethal disease in both AG129 and A129
mice but is more virulent in AG129 mice. TVP-376 is
lethal in AG129 mice only (Fig. 1a, 8a). Despite the mortality differences, weight loss data show that TVP-376
infection affects A129 mice similarly to AG129 mice, but
clinical infection in A129 is reversible because the lost
weight is ultimately regained. No differences were detected
between D2S10 and TVP-376 viraemia levels in A129 mice
or between TVP-376-infected AG129 and A129 mice
(Fig. 8c); thus, both viruses are capable of establishing
infection at a similar rate in both A129 and AG129, but
the clinical outcome varies.
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Journal of General Virology 96
Lethal DENV-4 model in AG129 mice using strain TVP-376
In conclusion, our study examines the differences in susceptibility of WT and immunocompromised mice to two
serologically and genetically distinct viruses, DENV-2
D2S10 and DENV-4 TVP-376. This study provided a platform to compare virulence and infectivity of two different
viruses in the same experimental setting, including mouse
strain, virus strain and dose. Understanding the similarities
and differences of the four DENVs in the same lethality
model will provide valuable information that is needed to
better evaluate candidate vaccines and antivirals, and
potentially provide information on the pathogenesis of
the four DENVs.
Fluorescence immunohistochemistry. Paraffin-embedded sec-
tions from 107.0 p.f.u.-TVP-376-infected AG129 mice were immunostained for non-structural protein 1 (NS1) using rabbit polyclonal
anti-DENV-2 NS1 (Genetex GTX124280) diluted 1 : 100, followed by
detection with goat anti-rabbit IgG F(ab’)2–Alexa Fluor 488 (Molecular Probes) diluted 1 : 200, and mounting with Vectashield
mounting medium containing DAPI (Vector Laboratories).
As controls, sections from mock-infected animals were stained with
anti-NS1, and infected sections were stained with rabbit IgG.
Complete blood count. Blood from AG129 mice inoculated with
medium or infected with 107.0 p.f.u. TVP-376 was collected into
EDTA anticoagulant tubes and analysed for blood counts using a
Hemavet 950TS (Drew Scientific).
METHODS
Cell culture. Monkey kidney Vero cells were maintained at 37 uC in
5 % CO2 in minimum essential medium (MEM) supplemented with
L -glutamine, non-essential amino acids, penicillin/streptomycin and
8 % bovine growth serum (BGS). C6/36 mosquito cells were maintained at 28 uC in MEM supplemented with L -glutamine, nonessential amino acids, penicillin/streptomycin, sodium pyruvate,
tryptose phosphate broth and 10 % FBS.
Virus. DENV-4 strain TVP-376 was amplified three times in C6/36
cells with 2 % FBS, without mouse passaging or adaptation. Generation of DENV-2 D2S10 has been described previously (Shresta et al.,
2006). Viruses were harvested and concentrated using a 50 kDa
molecular weight cut-off Amicon filter at 1500|g and 4 uC, for
20 min. Virus stocks were quantified by plaque titration assays in
Vero cells. Briefly, cells were infected with 10-fold virus dilutions for
30 min before overlay with MEM containing 2 % BGS/1 % agar and
incubated for 4 days at 37 uC. Plaques were counted 2 days after the
second overlay with MEM agar containing 2 % neutral red, and titres
are expressed as p.f.u. ml21. For some experimental titrations, focus
formation assays were performed with overlay of 0.8 % carboxymethyl cellulose; immunostaining was performed with the panmosquito-borne flavivirus mAb 4G2. Extensive comparisons yielded
equal results with plaque and focus assays.
Mouse infection. WT C57BL/6 mice (WT), C57BL/6 mice deficient
in mitochondrial antiviral signalling protein (Mavs 2/2), C57BL/6
mice deficient in IFN-a/b and IFN-c receptors (AGB6), 129/Sv mice
deficient in IFN-a/b receptor (A129), or 129/Sv mice deficient in IFNa/b and IFN-c receptors (AG129) were bred and maintained at the
University of Texas Medical Branch (UTMB). Experiments were
approved by the UTMB Institutional Animal Care and Use Committee and performed according to institutional guidelines. Infections
were performed in 6–8-week old mice via the intraperitoneal route.
Mice were weighed and visually monitored; mice exhibiting signs of
severe disease, weight loss below 80 % of original body weight, or
neurological dysfunction were euthanized. Animals surviving infection were monitored for at least 4 weeks post-infection. Lethal TVP376 infection of AG129 mice was characterized in mice inoculated
with 107 p.f.u. in two separate experiments. At 1, 2, 3 and 4 days p.i., a
group of three to five mice was sacrificed for downstream analysis,
including determination of viral loads, tissue sectioning, blood
counts, and cytokine analysis.
7.4
Mouse necropsy. Moribund AGB6 mice inoculated with 10
p.f.u.
TVP-376 were sacrificed at 3–4 days p.i., and AG129 mice inoculated
with 107.0 p.f.u. were sacrificed at 1–4 days p.i. Blood was collected by
cardiac puncture; liver, spleen, intestine and brain samples were collected
into pre-weighed tubes and homogenized; and virus was titrated. Mean
http://jgv.microbiologyresearch.org
limits of detection were as follows: serum 101.7 p.f.u., liver 102.2 p.f.u.,
spleen and large intestine 102.7 p.f.u, and brain 102.3 p.f.u.
Histology. At the time of necropsy, liver, spleen, intestine and brain
samples were harvested and immediately fixed in 10 % neutral-buffered formalin. Tissues were processed, and sections were stained with
H&E at the UTMB Research Histopathology Core Laboratory. Slides
from TVP-376-infected mouse tissues were analysed for histopathological changes, and spleen and liver slides were semi-quantitatively
scored from 22 to +2 relative to mock-infected controls, which were
assigned a score of 0. The parameters examined and histological
grading have been previously described and are listed in the legend of
Fig. 4 (Sarathy et al., 2015). Liver sections were also stained with PAS
to visualize the hepatocyte glycogen content (intense pink staining).
Cytokine analysis. Bio-Plex Pro Mouse Cytokine 23-plex (Bio-Rad)
was used to test cytokine levels in 15 ml serum according to the
manufacturer’s instructions. Mice were infected with 107.0 p.f.u. TVP376, and sera collected from AG129 mice 1–3 days p.i. were examined
and compared with same-day-matched mock-infected samples.
In vivo TNF-a neutralization. AG129 mice were inoculated with
107.0 p.f.u. TVP-376 (n510). At 1, 2 and 3 days p.i., mice were
administered 100 mg of either anti-TNF-a clone MP6-XT3
(eBioscience) (n55) or isotype control (n55), as described previously
(Shresta et al., 2006). Mice were monitored daily, and those exhibiting
severe disease were euthanized.
Virus neutralization. Focus reduction neutralization titration
(FRNT50) assays were performed on terminal bleed serum harvested
4 weeks post-infection. Twofold serial serum dilutions were incubated
with 50 p.f.u. infecting virus for 1 h and then used to infect Vero cells.
Titres were analysed using the log(inhibitor) versus normalized response–variable slope non-linear regression model to acquire the
FRNT50 and 95 % CI.
Statistical analyses. Neutralization curves were normalized using
Microsoft Excel. LD50 was calculated using the Spearman–Karber
method. All graphs and statistical analyses were determined using
Graphpad Prism v.6.0. Statistical significance is depicted in the figures
as follows: ****, Pv0.0001; ***, Pv0.001; **, Pv0.01; *, Pv0.05.
ACKNOWLEDGEMENTS
We thank Jeanon Smith for technical assistance. We thank Dr Michael
Gale (University of Washington School of Medicine) for providing
Mavs 2/2 mice. This work was supported in part by NIAID contract
N01 AI 30065 to A. D. T. B. and N. B. V. V. S. was supported by
NIAID T32 postdoctoral fellowship AI 7536-13.
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3047
V. V. Sarathy and others
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