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1
JNK pathway restricts DENV, ZIKV and CHIKV infection by activating complement 1
and apoptosis in mosquito salivary glands 2
3
4
5
Avisha Chowdhury,α, Cassandra M. Modahl1,α, Siok Thing Tan1, Benjamin Wong Wei 6
Xiang2, Dorothée Missé3, Thomas Vial2, R. Manjunatha Kini1,* and Julien Francis 7
Pompon2,3,* 8
9
10
11
1Department of Biological Sciences, National University of Singapore, Singapore 12
2Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 13
3MIVEGEC, IRD, CNRS, Univ. Montpellier, Montpellier, France 14
αAuthors contributed equally 15
*corresponding authors: [email protected]; [email protected] 16
17
18
Key words: dengue, Zika, chikungunya, Aedes aegypti, immunity, salivary glands, JNK 19
pathway 20
21
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 3, 2020. . https://doi.org/10.1101/2020.03.01.972026doi: bioRxiv preprint
2
22
Abstract (150 words) 23
Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. 24
However, there is a dearth of knowledge on SG immunity. Here, we characterized SG 25
immune response to dengue, Zika and chikungunya viruses using high-throughput 26
transcriptomics. The three viruses regulate components of Toll, IMD and JNK pathways. 27
However, silencing of Toll and IMD components showed variable effects on SG infection by 28
each virus. In contrast, regulation of JNK pathway produced consistent responses. Virus 29
infection increased with depletion of component Kayak and decreased with depletion of 30
negative regulator Puckered. Virus-induced JNK pathway regulates complement and 31
apoptosis in SGs via TEP20 and Dronc, respectively. Individual and co-silencing of these 32
genes demonstrate their antiviral effects and that both may function together. Co-silencing 33
either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. We 34
identified and characterized the broad antiviral function of JNK pathway in SGs, expanding 35
the immune arsenal that blocks arbovirus transmission. 36
37
38
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 3, 2020. . https://doi.org/10.1101/2020.03.01.972026doi: bioRxiv preprint
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Introduction 39
In recent decades, dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses have 40
emerged as global public health issues, with over 50% of the world population at risk for 41
infection (Kraemer et al., 2019). DENV and ZIKV belong to the Flavivirus genus 42
(Flaviviridae family), while CHIKV belongs to the Alphavirus genus (Togaviridae family). 43
DENV infects an estimated 390 million people yearly, causing a wide range of clinical 44
manifestations from mild fever to shock syndrome and fatal hemorrhage (Bhatt et al., 2013). 45
ZIKV recently emerged as an epidemic virus, infecting 1.5 million people over the past five 46
years (Hamel et al., 2016). Although ZIKV infection is mostly asymptomatic, it can result in 47
life-debilitating neurological disorders including Guillain-Barré syndrome in adults and 48
microcephaly in prenatally-infected newborns. CHIKV emerged as an epidemic virus in 49
2004, and has infected more than 6 million people (Silva & Dermody, 2017). It causes mild 50
fever but can result in musculoskeletal inflammation, leading to long-term polyarthralgia. 51
Amplified by urban growth, climate change and global travel, arboviral outbreaks are 52
unlikely to recede in the near future (Mayer et al., 2017). 53
DENV, ZIKV and CHIKV are primarily transmitted by Aedes aegypti mosquitoes. In 54
the absence of efficient vaccines (Wilder-Smith & Gubler, 2015) and curative drugs(Barrows 55
et al., 2018), targeting this common vector is the best available strategy to control the spread 56
of all three viruses. However, current vector control methods that rely on chemical 57
insecticides are not effective in preventing outbreaks (Wilder-Smith et al., 2017), partly due 58
to insecticide resistance (Liu, 2015). A novel strategy employing Wolbachia to reduce virus 59
transmission by mosquitoes has been deployed as a trial in several countries (Anders et al., 60
2018). However, its long-term efficacy may be compromised by converging bacteria and 61
virus evolution (Ritchie et al., 2018). Other promising approaches utilize genetic engineering 62
technology to develop refractory vector populations (Kistler et al., 2015). Mosquito innate 63
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immunity can drastically reduce virus transmission, and characterization of mosquito immune 64
pathways and mechanisms will aid in identifying gene candidates for transformation. 65
Immunity in midguts, the first organ to be infected following a blood meal, has been 66
extensively studied by using transcriptomics. DENV and ZIKV activate the Toll, Immune 67
Deficiency (IMD) and Janus Kinase (JAK)/Signal Transduction and Activators of 68
Transcription (STAT) immune pathways (Angleró-Rodríguez et al., 2017; Sim et al., 2013; 69
Souza-Neto et al., 2009; Xi et al., 2008). Selective gene silencing studies have demonstrated 70
the anti-DENV impact of Toll and JAK/STAT pathways. However, JAK/STAT transgenic 71
activation was not effective in reducing ZIKV and CHIKV infection (Jupatanakul et al., 72
2017). Immune effectors downstream of these pathways include antimicrobial peptides 73
(AMPs) and thioester containing proteins (TEPs) (Angleró-Rodríguez et al., 2017; 74
Jupatanakul et al., 2017; Ramirez & Dimopoulos, 2010; Sim et al., 2013; Souza-Neto et al., 75
2009). AMPs can have direct antiviral activity (Luplertlop et al., 2011), while TEPs that 76
belong to the complement system tag pathogens for lysis, phagocytosis or melanization 77
(Blandin, 2004; Xiao et al., 2014). Additionally, the RNA interference (RNAi) pathway 78
cleaves viral RNA genomes (Sanchez-Vargas et al., 2004). However, its impact against 79
arbovirus infection is uncertain (Olmo et al., 2018). In Drosophila melanogaster, the Jun-N-80
terminal Kinase (JNK) pathway regulates a range of biological functions including immunity 81
and apoptosis (Kockel et al., 2001). In Anopheles gambiae, the JNK pathway mediates an 82
anti-malaria response through complement activation (Garver et al., 2013). Currently, the 83
impact of JNK pathway on arbovirus infection remains unexplored. 84
Following midgut invasion, arboviruses propagate to remaining tissues, including 85
salivary glands (SGs), from where they are expectorated with saliva during subsequent bites. 86
Despite the critical role of SGs in transmission, only three studies have examined DENV-87
responsive differential gene expression in SGs. These studies revealed activation of the Toll 88
.CC-BY-NC-ND 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprintthis version posted March 3, 2020. . https://doi.org/10.1101/2020.03.01.972026doi: bioRxiv preprint
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and IMD pathways (Bonizzoni et al., 2012; Luplertlop et al., 2011; Sim et al., 2012) and 89
identified the anti-DENV functions of Cecropin (Luplertlop et al., 2011), putative Cystatin 90
and ankyrin-repeat proteins (Sim et al., 2012). Here, we characterized the SG immune 91
response to DENV, ZIKV and CHIKV. We performed the first high throughput RNA-92
sequencing (RNA-seq) in infected SGs and observed differentially expressed genes (DEGs) 93
related to immunity, apoptosis, blood-feeding and lipid metabolism. Using gene silencing, we 94
discovered that upregulated components of the Toll and IMD pathways had variable effects 95
against DENV, ZIKV and CHIKV. However, silencing of a JNK pathway upregulated 96
component increased, and silencing of a negative regulator decreased infection by the three 97
viruses in SGs. Further, we show that the JNK pathway is activated by all viruses and triggers 98
a cooperative complement and apoptosis response in SGs. This work identifies and 99
characterizes the JNK antiviral response that reduces DENV, ZIKV and CHIKV in A. aegypti 100
SGs. 101
102
Results 103
Transcriptome regulation by DENV, ZIKV and CHIKV in SGs 104
SGs were collected at 14 days post oral infection (dpi) with DENV and ZIKV, and at seven 105
dpi with CHIKV (Supplementary Figure 1) to account for variability between virus extrinsic 106
incubation periods (EIP) (Mbaika et al., 2016; Salazar et al., 2007). Differentially expressed 107
genes (DEGs) were calculated with DESeq2, edgeR and Cuffdiff 2, and showed little overlap 108
among the algorithms (Supplementary Figure 2). To validate DEGs and select which 109
software to use, we quantified the expression of 10 genes in a biological repeat with RT-110
qPCR and compared these values to the output from each algorithm. RNA-seq gene 111
expression obtained with DESeq2 correlated best with RT-qPCR values (DENV: r2 = 0.69; 112
ZIKV: r² = 0.81; CHIKV: r² = 0.79; Supplementary Figure 3), and only these DEGs are 113
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discussed. 114
The SG transcriptome was the most regulated by CHIKV infection (966 DEGs), 115
followed by ZIKV (396) and DENV (202) (Figure 1a; Supplementary Table 1). Only 19 116
DEGs were common amongst the three virus infections (Supplementary text), indicating a 117
virus-specific transcriptome response. Comparison between DEGs from the current DENV 118
infection and the three previous DENV studies with SGs collected at 14 dpi showed little 119
overlap among them (Supplementary Figure 4) (Bonizzoni et al., 2012; Luplertlop et al., 120
2011; Sim et al., 2012). However, despite technical variations (e.g. mosquito colony, virus 121
strain, transcriptomics technology), DEGs belonged to similar functional groups across the 122
studies. We observed that immunity, apoptosis, blood-feeding and lipid metabolism related 123
genes were highly regulated by DENV, ZIKV and CHIKV (Figure 1b; Supplementary text). 124
125
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126
Figure 1. DENV, ZIKV and CHIKV transcriptome regulation in A. aegypti salivary glands. 127
a, Venn diagram shows numbers of uniquely and commonly regulated differentially 128
expressed genes (DEGs) in DENV (green), ZIKV (red) and CHIKV (blue) infected salivary 129
glands. Colorless area shows total number of unchanged genes. Arrows indicate the direction 130
of regulation for the corresponding color code. b, Functional annotation of DEGs in DENV, 131
ZIKV or CHIKV infected salivary glands. APO, apoptosis; BF, blood feeding; CS, 132
cytoskeleton and structure; DIG, digestion; DIV, diverse functions; IMM, immunity; MET, 133
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metabolism; PROT, proteolysis; RSM, redox, stress and mitochondria; RTT, replication, 134
transcription and translation; TRP, transport; UNK, unknown functions. c, Transcriptomic 135
regulation of the IMD and JNK pathways in DENV, ZIKV and CHIKV infected salivary 136
glands. First column represents a scheme of the JNK and IMD pathways partially, modified 137
from (Sim et al., 2014). In the following columns, boxes indicate DEGs with AAEL number 138
below for DENV, ZIKV and CHIKV. Arrows indicate the direction of regulation. Pink-filled 139
boxes indicate genes regulated by more than one virus. Boxes with dotted line indicate DEGs 140
selected for functional studies. Dark and light shaded green areas differentiate IMD from 141
JNK pathways. 142
143
144
A high proportion of DEGs were related to immunity with DENV, ZIKV and CHIKV 145
modulating 20, 118 and 86 immune genes, respectively (Figure 1b; Supplementary Table 1). 146
Differential regulation of the RNAi, Toll, IMD, JAK/STAT and JNK pathways were 147
observed. The RNAi pathway is initiated when Dicer2 (Dcr2) recognizes and cleaves viral-148
derived dsRNA into small-interfering RNAs (siRNA) (Supplementary Figure 5). siRNAs are 149
loaded onto the RNA-induced silencing complex (RISC) that includes Argonaute2 (Ago2). 150
RISC unwinds siRNA to allow binding to complementary viral RNA fragments and cleavage 151
(Sanchez-Vargas et al., 2004). Strikingly, Dcr2 was upregulated by all viruses, while Ago2 152
was upregulated by DENV (Supplementary Figure 5), suggesting activation of RNAi. The 153
Toll pathway is triggered when extracellular pattern recognition receptors (PRR) (e.g. PGRP 154
and GNBP) bind to pathogen-derived ligands and activate a proteolytic cascade that leads to 155
activation of pro-Spätzle to Spätzle by Spätzle processing enzyme (SPE) (Supplementary 156
Figure 6) (DeLotto & DeLotto, 1998). Spätzle binding to transmembrane receptor Toll 157
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induces a cytoplasmic cascade that leads to nuclear translocation of NF-κB transcription 158
factor Rel1a to initiate effector gene transcriptions. Among PRRs, we observed that ZIKV 159
and CHIKV upregulated GNBPA1 and downregulated GNBPB6, while ZIKV also 160
upregulated PGRPS1 and CHIKV downregulated GNBP2 (Supplementary Figure 6). 161
Numerous serine proteases [e.g. CLIPs including Snake-likes (Snk-like) or Easter-likes (Est-162
like)] and serine protease inhibitors (Serpins) were upregulated by the viruses, except for Snk-163
like AAEL002273 and CLIPB41 downregulation by CHIKV, and SPE and CLIPB37 164
downregulation by DENV. SPE downregulation by DENV is reminiscent of downregulation 165
of Toll pathway components by subgenomic flaviviral RNA (sfRNA) in SGs (Pompon et al., 166
2017). The IMD pathway, similarly to Toll, is triggered by microbial ligand recognition by 167
PRRs, which activate PGRP-LC transmembrane protein (Figure 1c). In the ensuing 168
cytoplasmic cascade, NF-κB transcription factor Rel2 is phosphorylated by IKK2 and cleaved 169
by DREDD to induce its nuclear translocation. IMD pathway is repressed by Caspar (Sim et 170
al., 2014), an Ectoderm-expressed 4-containing module (Ect4) (Akhouayri et al., 2011) and 171
PGRP-LB (Gendrin et al., 2017). We observed an induction of IMD pathway by DENV and 172
ZIKV through PGRP-LB downregulation, and by CHIKV through Caspar downregulation 173
and IKK2 upregulation (Figure 1c). Ect4 upregulation by CHIKV may have controlled the 174
pathway activation. The JAK/STAT pathway is triggered by binding of Unpaired (Upd) to 175
transmembrane protein Domeless (Dome), which dimerizes and phosphorylates Hopscotch 176
(Hop) (Supplementary Figure 7) (Sim et al., 2014). The activated Dome/Hop complex 177
phosphorylates STATs, which upon dimerization translocate to nucleus and initiate 178
transcription. The phosphorylation of STATs is controlled by Suppressor of Cytokine 179
Signaling (SOCS) family of inhibitors (Callus & Mathey-Prevot, 2002). We observed an 180
upregulation of SOCS36E by CHIKV (Supplementary Figure 7), suggesting inhibition of the 181
pathway. The JNK pathway is triggered by Hemipterous (Hep) phosphorylation through 182
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tumor necrosis-associated factor 4 (Traf4) or Tak1, the latter being an IMD pathway 183
component (Figure 1c). Hep activates Basket (Bask) that phosphorylates c-Jun and Kayak 184
(Kay) transcription factors. Activated c-Jun and Kay dimerize to form the activator protein-1 185
(AP-1) complex, which translocates to nucleus and induces transcription. Puckered (Puc) 186
transcription is induced by the JNK pathway and represses Bask (Martín-Blanco et al., 1998), 187
acting as a feedback loop. Intriguingly, CHIKV upregulated most of the JNK pathway 188
components (i.e. Bask, c-Jun, Kay and Puc; Figure 1c), indicating activation of the pathway 189
in SGs. Furthermore, Traf4 upregulation by CHIKV suggested an IMD-independent 190
activation. 191
192
Upregulated components from JNK but not from Toll and IMD pathway reduce DENV, 193
ZIKV and CHIKV 194
To determine how immune response influences DENV, ZIKV and CHIKV multiplication in 195
SGs, we silenced nine previously uncharacterized immune genes that were upregulated by at 196
least one of the viruses (Table 1). They included four protease genes that initiate the Toll 197
pathway (CLIPB13A, CLIPB21, one of the Est-like, one of the Snk-like), two genes from the 198
cytoplasmic signaling of the IMD pathway (IKK2 and Ect4), one transcription factor gene of 199
the JNK pathway (Kay), and two putative immune genes - Galectin-5 (Gale5) and a juvenile 200
hormone induced gene (JHI). Gale5 shows antiviral function against O’nyong-nyong in A. 201
gambiae (Waldock et al., 2012), while juvenile hormone treatment regulates immune gene 202
expression in D. melanogaster (Flatt et al., 2008) and JHI was upregulated by all three 203
viruses in our study (Table 1). RNAi-mediated gene silencing efficacy ranged from 35-85 % 204
in SGs (Supplementary Figure 8a) and did not affect mosquito survival (Supplementary 205
Figure 9a). To bypass the midgut barrier, we intrathoracically inoculated mosquitoes with a 206
non-saturating inoculum of DENV, ZIKV or CHIKV, resulting in 70-80% infected SGs 207
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(Supplementary Figure 10). This permitted the evaluation of an increase or decrease in 208
infection upon gene silencing. At 10 days post inoculation, viral infection in SGs was 209
measured using two parameters, infection rate and infection intensity. Infection rate was 210
defined as the percentage of infected SGs out of 20 inoculated ones and represents 211
dissemination of intrathoracically injected viruses into SGs. Infection intensity was calculated 212
as viral genomic RNA (gRNA) copies per individual infected SGs and indicates virus 213
replication. Of note, the two infection parameters do not reflect isolated biological 214
phenomenon. For instance, a negative impact on infection intensity may lower infection rate 215
due to virus clearance. 216
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Table 1. Impact of immune response on DENV, ZIKV and CHIKV infection in salivary glands 217
1Induction in SGs as measured with RNA-seq. 2Determined by RNAi-mediated silencing studies in SGs. 218
219
220
221
222
223
Pathway Gene Virus Induced1 Function2
Proviral Antiviral
Dissemination Replication Dissemination Replication
Toll CLIPB13A
(AAEL003243)
DENV
ZIKV
CHIKV
CLIPB21
(AAEL001084)
DENV
ZIKV
CHIKV
Est-like
(AAEL012775)
DENV
ZIKV
CHIKV
Snk-like
(AAEL002273)
DENV
ZIKV
CHIKV
IMD IKK2
(AAEL012510)
DENV
ZIKV
CHIKV
Ect4
(AAEL014931)
DENV
ZIKV
CHIKV
JNK Kayak
(AAEL008953)
DENV
ZIKV
CHIKV
Putative
immune
JHI
(AAEL000515)
DENV
ZIKV
CHIKV
Gale5
(AAEL003844)
DENV
ZIKV
CHIKV
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Silencing of most of the immune genes had a virus-specific effect on infection 224
intensity and infection rate (Figure 2a-c and Table 1). Silencing of CLIPB13A increased 225
ZIKV infection rate, but decreased DENV and CHIKV infection rates. This suggests that 226
CLIPB13A hinders ZIKV dissemination and facilitates DENV and CHIKV dissemination into 227
SGs. However, it has a minor role in regulating virus replication in SGs. CLIPB21 silencing 228
increased ZIKV infection rate but decreased its infection intensity. Est-like silencing 229
decreased, whereas Snk-like silencing increased CHIKV infection rate. Overall, Toll pathway 230
upregulated components showed mostly a virus-specific impact on dissemination into SGs 231
(Figure 2a-c and Table 1). Silencing of IKK2 increased infection rate and decreased infection 232
intensity for ZIKV. Ect4 silencing increased DENV infection intensity and decreased CHIKV 233
infection rate. Overall, IMD pathway response appears both proviral and antiviral (Figure 2a-234
c and Table 1). Gale5 silencing decreased both ZIKV infection intensity and infection rate, 235
and enhanced CHIKV infection rate. Although JHI was upregulated by all viruses, it had no 236
effect on any infections (Figure 2a-c and Table 1). The silencing studies reflect a complex 237
interaction between transcriptomic response and antiviral functions (Table 1) with the notable 238
exception of Kay. Kay silencing increased infection rate to 100% for both ZIKV and CHIKV, 239
and increased infection intensities by 8.6-, 6.75- and 17.65-fold for DENV, ZIKV and 240
CHIKV, respectively (Figure 2a-c and Table 1). These results reveal a broad antiviral 241
function of the JNK pathway in SGs. To determine whether JNK pathway also restricts virus 242
in midguts, we orally infected Kay-silenced mosquitoes with ZIKV (Supplementary Figure 243
8b). At seven dpi, while infection rate was already saturated at 100% in the control, infection 244
intensity increased in midguts (Figure 2d). Overall, our functional studies discovered the 245
antiviral and proviral roles of several immune-related genes and established that JNK 246
pathway has a broad ubiquitous antiviral function. 247
248
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249
Figure 2. Kayak depletion but not Toll or IMD component depletion increases salivary 250
glands infection by DENV, ZIKV and CHIKV, and midgut infection by ZIKV. Four days 251
post dsRNA injection, mosquitoes were infected by intrathoracic inoculation with DENV, 252
ZIKV or CHIKV, or by oral feeding on ZIKV. Viral genomic RNA (gRNA) was quantified 253
at 10 days post inoculation in salivary glands and 7 days post oral infection in midguts. a-c, 254
Effect of immune-related gene silencing on gRNA copies and infection rate in salivary glands 255
infected with (a) DENV, (b) ZIKV, and (c) CHIKV. d, Impact of Kayak silencing on gRNA 256
number and infection rate in ZIKV-infected midgut. Bars show geometric mean ± 95% C.I. 257
from 20 individual pairs of salivary glands or 25 individual midguts. Each dot represents one 258
sample. dsCtrl, dsRNA against LacZ; dsCLIPB13A, dsRNA against CLIP domain serine 259
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protease B13A; dsCLIPB21, dsRNA against CLIP domain serine protease B21; dsEst-like, 260
dsRNA against Easter-like; dsSnk-like, dsRNA against Snake-like; dsIKK2, dsRNA against 261
Inhibitor of nuclear factor kappa-B kinase; dsKay, dsRNA against Kayak; dsEct4, dsRNA 262
against Ectoderm expressed-4; dsJHI, dsRNA against Juvenile hormone inducible; dsGale5, 263
dsRNA against Galectin 5. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001, 264
determined by post hoc Dunnett’s with dsCtrl, unpaired t-test or Z-test for infection rate. 265
266
JNK pathway is induced by DENV, ZIKV and CHIKV and reduces infection in SGs 267
SG transcriptomics showed that Kay was induced by CHIKV at seven dpi, but not by DENV 268
or ZIKV at 14 dpi (Figure 1c). To test whether JNK pathway is activated by the three viruses, 269
we monitored the kinetics of Kay expression in SGs after oral infection with either of the 270
three viruses. Kay was similarly induced by DENV, ZIKV and CHIKV at three and seven 271
dpi, but not at 14 dpi (Figure 3a), corroborating the RNA-seq data. We quantified Puc 272
expression, which is induced as a negative regulator (Martín-Blanco et al., 1998). Although 273
not regulated at three and 14 dpi, Puc expression was increased at seven dpi for all three 274
viruses (Figure 3b). We then tested the impact of Puc silencing in ZIKV-inoculated 275
mosquitoes (Supplementary Figure 8c). Puc silencing did not alter mosquito survival 276
(Supplementary Figure 9b). Although SG infection rate was not affected, SG infection 277
intensity was decreased by 8.85-fold at 10 days post inoculation (Figure 3c). Altogether our 278
data demonstrate that the JNK pathway is induced by DENV, ZIKV and CHIKV at a time 279
that corresponds to the onset of infection in SGs (Salazar et al., 2007) and further support the 280
JNK antiviral function. 281
282
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283
Figure 3. Kayak and Puckered expressions are induced by DENV, ZIKV and CHIKV 284
infection, and Puckered depletion restricts ZIKV infection in salivary glands. a, Kayak (Kay) 285
and b, Puckered (Puc) expressions in salivary glands at 3, 7 and 14 days post oral infection 286
with DENV, ZIKV and CHIKV. Gene expression was quantified in pools of 10 salivary 287
glands. Actin expression was used for normalization. Bars show arithmetic means ± s.e.m. 288
from three biological replicates. c, Effect of Puc depletion on infection in salivary glands at 7 289
days post ZIKV inoculation. Four days prior infection, mosquitoes were injected with 290
dsRNA. Bars show geometric means ± 95% C.I. from 20 individual salivary glands. Each dot 291
represents one sample. *, p-value < 0.05, as determined by Dunnett’s test within time points 292
with mock infection as control (a, b) or unpaired t-test (c). 293
294
The JNK pathway mediates an antiviral complement and apoptosis response in SGs 295
JNK pathway can regulate complement system (Garver et al., 2013), apoptosis (McEwen, 296
2005) and autophagy (Wu et al., 2009). To determine whether JNK pathway induces these 297
functions in SGs, we monitored the impact of Kay silencing on expressions of four TEPs 298
(TEP20, TEP24, TEP15 and TEP2), two pro-apoptotic (Caspase8 and Dronc) and two 299
autophagy-related (ATG14 and ATG18A) genes at 10 dpi with ZIKV. All these genes were 300
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upregulated by infection in the transcriptomic data (Figure 1c; Supplementary Table 1). 301
Control mosquitoes were injected with control dsRNA (dsCtrl). Kay silencing significantly 302
reduced expressions of TEP20 and Dronc, and moderately reduced TEP15 and TEP24 303
(Figure 4a). TEP2 expression was increased and the two ATGs and Caspase 8 were 304
unaffected. These results suggest that upon infection the JNK pathway induces the 305
complement system through TEPs, and apoptosis through Dronc, but not Caspase8. We then 306
tested the antiviral function of TEP20 and Dronc in SGs by challenging either TEP20- or 307
Dronc-silenced mosquitoes (Supplementary Figure 8d) by ZIKV intrathoracic inoculation. 308
Similar to Kay-silencing, TEP20- and Dronc-silencing increased SG infection intensity by 309
21-fold to 2.6 x 106 gRNA and by 12-fold to 1.3 x 106 gRNA, respectively (Figure 4b). Since 310
both complement and apoptosis can interact for cell clearance (Fishelson, 2001), we 311
determined whether TEP20 and Dronc act in the same antiviral pathway by evaluating their 312
synergistic effect when both were co-silenced (Supplementary Figure 8e). We did not 313
observe a clear difference in SG infection between TEP20 and Dronc individual or co-314
silencing (Figure 4c,d). As the infection conditions did not saturate the infection intensity 315
(higher inoculum resulted in 108 gRNA per infected SG; Supplementary Figure 10), the lack 316
of synergism when TEP20 and Dronc are co-silenced suggests that TEP20 and Dronc 317
function in the same antiviral pathway. To show that TEP20 and Dronc mediate the JNK 318
antiviral response in SGs, we induced JNK pathway by silencing of Puc and co-silenced 319
TEP20 or Dronc before ZIKV inoculation. Control mosquitoes were injected with the same 320
quantity of dsCtrl or dsRNA against Puc. While infection intensity decreased upon Puc 321
silencing, co-silencing of Puc with TEP20 or Dronc restored viral gRNA copies to the level 322
in dsCtrl-injected SGs (Figure 4d). Altogether, these results demonstrate that the JNK 323
pathway is induced by DENV, ZIKV or CHIKV in SGs and eliminates viruses through the 324
complement system and apoptosis. 325
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326
Figure 4. Virus-induced JNK pathway activates an antiviral response through complement 327
and apoptosis. a, Impact of Kayak depletion on expression of genes related to complement 328
(TEP20, TEP24, TEP15 and TEP2), apoptosis (Caspase8 and Dronc) and autophagy (ATG14 329
and ATG18A) at 10 days post ZIKV oral infection. Gene expression was quantified in pools 330
of 10 SGs. Actin expression was used for normalization. Bars show arithmetic means ± s.e.m. 331
from three biological replicates. b-d, Effect of depletion of (b) Dronc or TEP20 alone, (c) 332
Dronc and TEP20 simultaneously, and (d) Puc alone, or Puc and Dronc simultaneously, or 333
Puc and TEP20 simultaneously on SG infection at 7 days post ZIKV inoculation. Four days 334
prior infection, mosquitoes were injected with dsRNA. Bars show geometric means ± 95% 335
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C.I. from 20 individual salivary glands. Each dot represents one sample. TEP, Thioester-336
containing protein; ATG, Autophagy related gene; Kay, Kayak; Puc, Puckered; Ctrl, control. 337
*, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001, as determined by unpaired t-test 338
(a) or Dunnett’s test with dsCtrl as control (b-d). 339
340
Discussion 341
Dengue, Zika and chikungunya are widespread mosquito-borne diseases primarily 342
transmitted by A. aegypti. Disease mitigation through insecticide-based vector control has 343
mostly remained ineffective to prevent outbreaks (Wilder-Smith & Gubler, 2015). Currently, 344
insecticide-free vector-control strategies are being extensively investigated. The improved 345
understanding of the vector-virus interaction at the molecular level proposed here paves the 346
way to manipulate mosquito biology to make them refractory towards arboviruses. This study 347
discovers the antiviral function of the JNK pathway in SGs against three major arboviruses, 348
DENV, ZIKV and CHIKV belonging to two families. Further, we determine that the antiviral 349
response is mediated through a combined action of complement system and apoptosis. 350
Several studies have functionally characterized the immune response in A. aegypti 351
midgut, the first barrier in the mosquito body, determining the antiviral function of Toll, IMD 352
and JAKSTAT pathways (Angleró-Rodríguez et al., 2017; Bonizzoni et al., 2012; Dong et 353
al., 2017; Sim et al., 2013). However, only a couple of studies tested the impact of SG (exit 354
barrier) immune response (Luplertlop et al., 2011; Sim et al., 2012). Luplertlop et al., 2011 355
used Digital Gene Expression tag profiling to quantify the impact of DENV infection in SGs. 356
They reported overexpression of Toll pathway components, but did not test their functions. 357
Instead, they characterized the most abundant protein, an AMP from the Cecropin family, and 358
revealed its broad antiviral function in vitro. This Cecropin was not regulated in our 359
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transcriptomic analysis, although two other Cecropins (Cecropin A and G) were differentially 360
modulated by the different viruses. Sim et al., 2012 used microarrays to identify SG-specific 361
transcripts as compared to carcasses. An enrichment in immune-related genes suggested the 362
ability to mount an immune response. Further, the same authors determined the transcriptome 363
upon DENV infection. Similar to our data, they reveal a high regulation of serine proteases 364
that could initiate the different immune pathways or play a role in blood feeding when 365
secreted. While they did not functionally test the immune pathways, Sim et al. revealed the 366
antiviral and proviral functions of three DENV-upregulated genes. This supports the complex 367
interaction between gene regulation and function that we also observed. In our study, we 368
functionally tested for the first time the impact of upregulated components from Toll, IMD 369
and JNK pathways on DENV, ZIKV and CHIKV in SGs. JAK/STAT pathway was not 370
activated at the studied time-points. The lack of impact against DENV, ZIKV and CHIKV 371
when silencing the components of Toll and IMD pathways may not reflect the antiviral 372
capabilities of these pathways. Indeed, it is possible that a complete shutdown of the 373
signaling cascades increases virus infection. Importantly, we discovered the antiviral impact 374
of the JNK pathway against DENV, ZIKV and CHIKV in SGs. 375
JNK pathway activation by DENV, ZIKV and CHIKV in SGs occurred early during 376
infection at 3 dpi. This time corresponds to the onset of SG infection (Salazar et al., 2007). 377
Such an early induction may affect virus dissemination to SGs as reflected in ZIKV and 378
CHIKV infection rates. The JNK pathway can be induced either through IMD-mediated 379
pathogen recognition or oxidative stress. Tak1 activates the IMD and JNK pathways through 380
IKK2 and Hep, respectively (Takatsu et al., 2000). Since Tak1-mediated JNK activation is 381
transient, lasting less than one hour (Park, 2004), the observed JNK activation (lasting 3-7 382
dpi) is probably due to an IMD-independent activation. Upon oxidative stress, the JNK 383
pathway is induced through p53 upregulation of Traf4 that then phosphorylates Hep (Lu et 384
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al., 2017; Myers et al., 2018; Sax & El-Deiry, 2003). Activated JNK pathway then induces 385
other oxidative stress-associated genes, such as FoxO and κ-B Ras (AAEL003817) (Essers et 386
al., 2004). In our transcriptomic data, we reported the upregulation of several oxidative 387
stress-associated genes upon infection including p53, Traf4, FoxO, CYPs and κ-B Ras (Table 388
S1). These results suggest that JNK pathway is activated in SGs as a result of infection-389
induced oxidative stress. 390
Using in vivo functional studies, we showed that the JNK antiviral function depends 391
on complement system and apoptosis inductions. The complement system is activated when 392
TEPs bind to pathogens and trigger lysis, phagocytosis (Levashina et al., 2001) or even AMP 393
production (Xiao et al., 2014). TEPs such as TEP15 and AaMCR can restrict DENV in 394
mosquitoes (Cheng et al., 2011; Xiao et al., 2014), although TEP22 does not (Jupatanakul et 395
al., 2017). Apoptosis was previously reported in SGs infected with different flaviviruses and 396
alphaviruses (Day et al., 1966; Girard et al., 2005; Kelly et al., 2012). Apoptosis antiviral 397
function in mosquitoes is supported by two observations. Firstly, an arbovirus with an 398
apoptosis-inducing transgene was selected out during mosquito infection (O’Neill et al., 399
2015), and secondly, there is an association between the ability to induce apoptosis and 400
colony refractoriness in different virus-mosquito systems (Clem, 2016; Ocampo et al., 2013; 401
Vaidyanathan & Scott, 2006). These indicate that apoptosis can define vector competence, 402
emphasizing its importance as a target to control transmission. In our study, we reported the 403
antiviral functions of a complement system component, TEP20, and an apoptotic component, 404
Dronc, extending our understanding of the two mechanisms in SGs. Based on a lack of 405
synergistic effect when both TEP20 and Dronc were silenced, we propose that complement 406
system and apoptosis function in the same cascade to reduce virus infection. The complement 407
system could mediate apoptotic cell clearance as in mammals (Fishelson, 2001) and in testes 408
of A. gambiae (Pompon & Levashina, 2015). Alternatively, apoptosis-triggered nitration of 409
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22
virus surface could be required to direct TEP binding, as in Anopheles mosquitoes infected 410
with parasites (Kumar et al., 2010; Oliveira et al., 2012). 411
A recent study established a negative association between the presence of efficient 412
Plasmodium-killing immune response in mosquitoes and epidemics in Africa, confirming the 413
long-suspected impact of mosquito immunity on epidemiology for arthropod-borne diseases 414
(Gildenhard et al., 2019). Upon close inspection of field-derived A. aegypti colony 415
transcriptomes (Sim et al., 2013), we found that DENV-refractory colonies expressed a higher 416
level of Kay, c-Jun and TEP20. This suggests that variation in vector competence among these 417
colonies is partially related to JNK pathway. Consistent with our study, this points to a role of 418
JNK pathway in determining the arbovirus transmission dynamics in the field. Because JNK 419
pathway is sensitive to various microbes (Guntermann & Foley, 2011), differential activation 420
by distinct microbes present in natural habitats may also represent a trigger that influences 421
transmission. 422
Development of transgenic refractory mosquitoes have gained prominence in 423
preventing arboviral transmission (Wilke et al., 2018). Engineered overexpression of a 424
JAK/STAT activator in mosquitoes reduced DENV propagation and established its proof-of-425
concept (Jupatanakul et al., 2017). However, JAK/STAT is ineffective against ZIKV and 426
CHIKV. To our knowledge, no promising candidates have been identified to be antiviral 427
against DENV, ZIKV and CHIKV. In this context, our work reveals that the JNK pathway 428
components could be harnessed to develop effective transmission blocking tools against a 429
broad range of arboviruses. 430
431
Acknowledgments 432
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We thank the Professor Eng Eong Ooi from Duke-NUS Medical School, for providing the 433
DENV and ZIKV stock, and Professor Lisa Ng from Singapore Immunology Network (SIgN, 434
A*STAR, Singapore), for providing the CHIKV stock. This work was funded by the Tier-3 435
grant from the Ministry of Education, Singapore (MOE 2015-T3-1-003) and the Duke-NUS 436
Signature Research Programme funded by the Agency for Science, Technology and Research 437
(A*STAR), Singapore, and the Ministry of Health, Singapore. 438
439
Author Contributions 440
J.F.P. and R.M.K. designed the project. A.C., S.T.T. and B.W. performed the experiments. 441
C.M.M. performed bioinformatics analysis. A.C., C.M.M., D.M., T.V., R.M.K. and J.F.P. 442
analyzed the data. A.C. and C.M.M. made the figures. A.C., C.M.M., R.M.K. and J.F.P. 443
wrote the manuscript. All authors reviewed, critiqued and provided comments on the text. 444
445
Declarations of Interests 446
The authors declare no conflict of interest. 447
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Material and Methods 448
Mosquitoes 449
Aedes aegypti mosquitoes were collected in Singapore in 2010. Eggs were hatched in MilliQ 450
water and larvae were fed with a mixture of fish food (TetraMin fish flakes), yeast and liver 451
powder (MP Biomedicals). Adults were reared in cages (Bioquip) supplemented with 10% 452
sucrose and water. The insectary was held at 28°C and 50% relative humidity with a 12h:12h 453
light:dark cycle. 454
455
Virus isolates 456
The dengue virus serotype 2 PVP110 (DENV) was isolated from an EDEN cohort patient in 457
Singapore in 2008 (Christenbury et al., 2010). The Zika virus Paraiba_01/2015 (ZIKV) was 458
isolated from a febrile female in the state of Paraiba, Brazil in 2015 (Tsetsarkin et al., 2016). 459
The chikungunya virus SGP011 (CHIKV) was isolated from a patient at the National 460
University Hospital in Singapore (Her et al., 2010). DENV and ZIKV isolates were 461
propagated in C6/36 and CHIKV in Vero cell line. Virus stocks were titered with BHK-21 462
cell plaque assay as previously described (Manokaran et al., 2015), aliquoted and stored at -463
80°C. 464
465
Oral infection 466
Three-to-five day-old female mosquitoes were starved for 24 h and offered an infectious 467
blood meal containing 40% volume of washed erythrocytes from serum pathogen free (SPF) 468
pig’s blood (PWG Genetics), 5% 10 mM ATP (Thermo Scientific), 5% human serum 469
(Sigma) and 50% virus solution in RPMI media (Gibco). Mosquitoes were left to feed for 1.5 470
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h using Hemotek membrane feeder system (Discovery Workshops) covered with porcine 471
intestine membrane (sausage casing). The virus titers in blood meals were 2 x 107 pfu/ml for 472
DENV, 6 x 106 pfu/ml for ZIKV, and 1.5 x 108 pfu/ml for CHIKV, and validated in plaque 473
assay using BHK-21 cells. Control mosquitoes were fed with the same blood meal 474
composition except for virus. Fully engorged females were selected and kept in a cage with 475
ad libitum access to a 10% sucrose solution in an incubation chamber with conditions similar 476
to insect rearing. 477
478
Virus inoculation 479
Mosquitoes were cold anesthetized and intrathoracically inoculated with either DENV, ZIKV 480
or CHIKV using Nanoject-II (Drummond). The same volume of RPMI media (ThermoFisher 481
Scientific) was injected as control. Functional studies were conducted by inoculating 0.1 pfu 482
of DENV, 0.01 pfu of ZIKV and 0.2 pfu of CHIKV. 483
484
SG collection, library preparation and RNA-sequencing 485
SGs from DENV- and ZIKV-orally infected mosquitoes were dissected at 14 dpi. Those from 486
CHIKV-orally infected mosquitoes were dissected at 7 dpi. Controls for CHIKV were 487
dissected at 7 days post feeding on a non-infectious blood meal, and at 14 days post feeding 488
for DENV and ZIKV. Inoculum resulted in 100% infected SGs (Supplementary Figure 1). 489
Fifty pairs of SGs per condition were homogenized using a bead mill homogenizer (FastPrep-490
24, MP Biomedicals). Total RNA was extracted using E.Z.N.A Total RNA kit I (OMEGA 491
Bio-Tek). RNA-seq libraries were prepared using True-Seq Stranded Total RNA with Ribo-492
Zero Gold kit (Illumina), according to manufacturer’s instructions. Following quantification 493
by RT-qPCR using KAPA Library Quantification Kit (KAPA Biosystems), libraries were 494
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pooled in equimolar concentrations for cluster generation on cBOT system (Illumina) and 495
sequenced (150 bp pair-end) on a HiSeq 3000 instrument (Illumina) at the Duke-NUS 496
Genome Biology Facility, according to manufacturer’s protocols. Two repeats per condition 497
were processed. The need for more than two repeats was mitigated by using a large number 498
of tissues from different mosquitoes in each repeat. Raw sequencing reads from RNA-seq 499
libraries are available online under NCBI accessions: SRR8921123-8921132. 500
501
RNA-seq data processing and identification of differentially expressed genes (DEGs) 502
Reads were quality checked with FastQC (www.bioinformatics.babraham.ac.uk) to confirm 503
that adapter sequences and low-quality reads (Phred+33 score < 20) had been removed. The 504
reads were then aligned against the A. aegypti genome [VectorBase(Giraldo-Calderón et al., 505
2015)] AaegL3.3) using TopHat v2.1.0 (Kim et al., 2013) with parameters –N 6 –read-gap-506
length 6 –read-edit-dist. 6 set to account for regional genetic variation between the RNA-seq 507
and genome. SAM tools v0.1.19 (Li et al., 2009) and HTSeq v0.6.0 (Anders et al., 2015) 508
were used to format and produce count files for gene expression analysis. DEGs were 509
identified using DESeq2 (Love et al., 2014) with at least 1.4-fold change between control and 510
infected conditions, an adjusted False Discovery Rate (FDR) of 0.05. edgeR (Robinson et al., 511
2010) and Cuffdiff 2 (Trapnell et al., 2013) (following the Cufflinks pipeline (Trapnell et al., 512
2012)) were also used to identify DEGs following the same criteria. 513
514
Gene annotations 515
DEGs were annotated through several pipelines. Predicted gene protein sequences were 516
searched against the NCBI nr database (accessed July 2017) (BLASTp; e-value 1.0E-5 and 517
word size 3), VectorBase (Giraldo-Calderón et al., 2015) Aedes peptide database 518
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27
(downloaded July 2017) with BLAST+ (Camacho et al., 2009) (BLASTp; e-value 1.0E-5 519
threshold and word size 2), and FlyBase (Gramates et al., 2017) D. melanogaster peptide 520
database (downloaded August 2017) with BLAST+ (BLASTp; e-value 1.0E-5 threshold and 521
word size 2) for identification of orthologues. Gene ontology terms were assigned in 522
BLAST2GO (Conesa et al., 2005) (program default parameters). Signal peptides were 523
identified using SignalP v4.1 (Petersen et al., 2011). Functional annotations were also 524
assigned based on literature. 525
526
Double-stranded mediated RNAi 527
Mosquito cDNA was used to amplify dsRNA targets with gene specific primers tagged with 528
T7 promoter as detailed in Supplementary Table 2. The amplified products were in vitro 529
transcribed with T7 Scribe kit (Cellscript). dsRNAs were annealed by heating to 95°C and 530
slowly cooling down to 4°C using a thermocycler. Three to five-day-old adult female 531
mosquitoes were cold-anesthetized and intrathoracically injected with 2 or 4 µg of dsRNA 532
using Nanoject II. The same quantity of dsRNA against the bacterial gene LacZ was injected 533
as control (dsCtrl). Validation of gene silencing was conducted 4 days post injection by 534
pooling 10 SGs or 5 midguts. 535
536
Gene expression quantification using RT-qPCR 537
Total RNA was extracted from 10 SGs or 5 midguts using E.Z.N.A. Total RNA kit I, DNAse 538
treated using Turbo DNA-free kit (Thermo Fisher Scientific), and reverse transcribed using 539
iScript cDNA synthesis kit (Biorad). Gene expression was quantified using qPCR with 540
SensiFast Sybr no-rox kit (Bioline) and gene specific primers detailed in Supplementary 541
Table 2, 3 and 4. Actin expression was used for normalization. The reactions were performed 542
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using the following cycle conditions: an initial 95°C for 10 min, followed by 40 cycles of 543
95°C for 5 s, 60°C for 20 s and ending with a melting curve analysis. The delta delta method 544
was used to calculate relative fold changes. 545
546
Quantification of virus genome RNA (gRNA) copies using RT-qPCR 547
Individual pairs of either SGs or midguts were homogenized with a bead Mill Homogenizer 548
in 350 µl of TRK lysis buffer (E.Z. N. A Total RNA kit I). Total RNA was extracted using 549
the RNA extraction kit and reverse-transcribed using iScript cDNA synthesis kit. DENV 550
gRNA copies were quantified by RT-qPCR using i-Taq one step universal probes kit 551
(BioRad) and ZIKV and CHIKV gRNA copies with i-Taq one step universal sybr kit 552
(BioRad) with primers detailed in Supplementary Table 5. Amplification was run on CFX96 553
Touch Real-Time PCR Detection System (BioRad) with the following thermal profile: 50°C 554
for 10 min, 95 °C for 1 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 15 s. A melt-555
curve analysis was added for Sybr qPCR. 556
Absolute quantification of gRNA was obtained by generating a standard curve for 557
each virus target. Viral cDNA was used to amplify qPCR target using qPCR primers with T7-558
tagged forward primer. RNA fragments were generated with T7-Scribe kit and RNA 559
concentration calculated by Nanodrop was used to estimate concentration of RNA fragments. 560
Ten-time serial dilutions were quantified with RT-qPCR and used to generate absolute 561
standard equations. Three standard dilutions were used in each subsequent RT-qPCR plate to 562
adjust for inter-plate variation. 563
564
Statistical analyses and software used 565
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One-way ANOVA and post-hoc Dunnett’s test or unpaired T-test were used to test 566
differences in gene expression and log10-transformed gRNA copies. These analyses were 567
done using GraphPad Prism 5. Z-score was used to test differences in infection rate with 568
www.socscistatistics.com/tests/ztest/. Standard error for sample proportion was calculated 569
with www.easycalculation.com. 570
571
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