Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti.

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.

Introduction

Mosquito-borne viruses pose a significant global health threat, and this threat is increased by dynamic ecological and human factors. Global warming and urbanization have permitted mosquitoes and arboviruses to spread into regions that previously lacked mosquito infection and mosquito-to-human virus transmission [1,2]. This occurred during the 2015–17 Zika virus (ZIKV) Western hemisphere epidemic that originated in South America and spread into North America, resulting in 538,451 suspected cases, 223,477 confirmed cases, and 3,720 congenital syndrome cases [3,4]. Subsequent outbreaks have followed that establishes ZIKV as an active pathogen of concern that requires intervention [5]. Current efforts have focused on strategies to reduce virus transmission to and from the mosquito vector, including the use of insecticides and biological/genetic manipulation of primary vector species. The introduction of the bacterial symbiont Wolbachia to reduce flavivirus infection in the major arbovirus vector species Aedes aegypti [6-8] and the release of genetically modified individuals to reduce transmission-competent progeny of this species [9] have been included among the latter strategies. There is evidence to suggest that Wolbachia, while effective in reducing ZIKV and dengue virus (DENV) infection in targeted species may inadvertently enhance replication of West Nile virus (WNV) [10]. It is also not known how effective or advantageous genetically modified mosquito populations are compared to wild type populations or to other various viruses [11,12]. Because of these challenges, additional strategies to reduce vector transmission of these important viral pathogens are critically needed.

As an alternative strategy, it may be possible to reduce or block arborvirus transmission through mosquito-targeted delivery of bioactive small molecules at attractive sugar bait stations, a modification of the successful delivery of toxic baits for mosquito control [13]. To this end, it is necessary to identify druggable mosquito antiviral effectors and their upstream regulatory factors. The insulin/insulin-like growth factor signaling (IIS) cascade regulates RNA interference (RNAi) and JAK/STAT antiviral immunity against West Nile virus (WNV), dengue virus (DENV), and ZIKV [14,15]. In Drosophila melanogaster, the IIS-dependent transcription factor forkhead box O (FOXO) induces expression of RNAi transcripts AGO2 and Dicer2 [16]. We demonstrated that ingestion of exogenous insulin reduced expression of these RNAi components in WNV-infected Culex quinquefasciatus [14] and that manipulation of IIS-dependent extracellular-signal regulated kinases (ERK) activation reduced WNV infection in this mosquito host [14]. Further, insulin treatment suppressed the activation of RNAi, while activating ERK-dependent JAK/STAT induction of unpaired (upd) ligands to control WNV replication in vitro and in vivo [14]. Previous studies established that both JAK/STAT and RNAi antiviral pathways are independently involved in arthropod antiviral immunity to ZIKV [17-20]. To date, however, no mechanism(s) have been established whereby both antiviral pathways are induced simultaneously in response to arthropod infection.

In this study, we repurposed small molecules that target IIS pathway proteins to induce simultaneous activation of RNAi and JAK/STAT signaling in Ae. aegypti. Specifically, we used the potent insulin mimetic demethylasterriquinone B1 (DMAQ-B1), an activating ligand of the insulin receptor (InR) [21] and Protein kinase B (AKT) inhibitor VIII, which reduces AKT phosphorylation [22] (). Small molecule treatment induced activation of JAK/STAT via ERK and blocked inhibition of RNAi via the AKT/FOXO signaling axis. Combined treatment with DMAQ-B1 and AKT inhibitor VIII significantly lowered ZIKV titers in Ae. aegypti cells and adult female mosquitoes relative to single treatment and vehicle control. Combined treatment also additively reduced salivary gland virus titers, a surrogate measure of reduced transmission efficacy [23,24]. Accordingly, we argue that activation of both antiviral pathways resulted in enhanced defenses that lowered viral titers to non-detectable levels. This work demonstrates the feasibility of strategically targeting mosquito immunity via IIS as a means of reducing a clinically relevant strain of ZIKV infection and transmission at the vector level.

DMAQ-B1 and AKT inhibitor VIII activated RNAi and JAK/STAT in vitro.

(A) Insulin/insulin-like growth factor I signaling (IIS) pathway schematic and proposed effect of DMAQ-B1 and AKT inhibitor VIII on downstream transcription factor activity. (B-E) Aag2 cells were treated with vehicle (DMSO), 1 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs for 24 h. Phosphorylation of AKT, FOXO, and ERK were measured by western blot and phosphorylation was quantified for (B) P-AKT, (C) P-FOXO, and (D) P-ERK by densitometry and normalized to actin (*p < 0.05, One-way ANOVA with Tukey’s test correction for multiple comparisons). (F) P-FOXO and P-ERK abundance and localization were visualized in DAPI-stained Aag2 cells by immunofluorescence microscopy. (G-H) Protein localization of (F) P-FOXO and (G) P-ERK was quantified in microscopy images to evaluate whether fluorescent-tagged proteins was cytosolic or nuclear within individual cells by manually counting cells in images (***p<0.001, Two-way ANOVA with Tukey’s correction). (I-J) Induction of RNAi and JAK/STAT signaling was evaluated as transcript levels of (I) AGO2 and (J) vir-1 by qRT-PCR (*p < 0.05; **p<0.01, Unpaired t test with Welch’s correction for multiple comparisons). (K) Aag2 cells that received vehicle, individual, or combined drug treatment for 24 h were infected with ZIKV (MOI 0.01) and supernatant was collected at 1 and 3 d p.i. Supernatant virus was titered by standard plaque assay (****p<0.0001, Two-way ANOVA with Tukey’s correction). Closed circles represent individual replicates. Horizontal bars represent mean and error bars represent SD. Results are representative of triplicate independent experiments.

Results

DMAQ-B1 and AKT inhibitor VIII activated Aedes aegypti insulin and antiviral signaling pathways

Since the JAK/STAT and RNAi antiviral pathways are linked to IIS, we sought to test the activity of small molecules against phosphorylation of key IIS protein targets and activity of these antiviral pathways. Given that phosphorylation of AKT and ERK correlate with activation of IIS and JAK/STAT signaling [14,25], respectively, and that FOXO phosphorylation is consistent with suppression of RNAi [16,26,27], we used these readouts to evaluate the efficacy of DMAQ-B1 and AKT inhibitor VIII control of RNAi and JAK/STAT.

Protein lysates from Ae. aegypti Aag2 cells treated with 1% DMSO vehicle, 1 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or these combined drug treatment for 24 hours were analyzed by western blot for phosphorylation of AKT, FOXO, and ERK (). Drug concentrations were based on prior toxicity analysis (). DMAQ-B1 treatment was associated with the highest levels of AKT and FOXO phosphorylation; this phosphorylation was significantly reduced when combined with AKT inhibitor VIII (). Single drug and combined drug treatments were associated with increased ERK phosphorylation relative to vehicle control (). We validated these findings by immunofluorescence microscopy of 24 h treated cells probed for phospho-FOXO (P-FOXO) and P-ERK. Consistent with western blot analyses, we observed increased P-FOXO only in the DMAQ-B1-treated cells and P-ERK in both individual and combined-treated cells (). Further, P-FOXO localization was primarily cytosolic () in DMAQ-B1 treated cells and P-ERK localization was primarily nuclear () in AKT inhibitor and combined treated cells, confirming that the transcription factors involved in RNAi and JAK/STAT induction are both nuclear and transcriptionally active under the expected treatment conditions. We also observed increased transcript expression of AGO2 and virus-induced RNA-1 (vir-1) which are indicative of RNAi and JAK/STAT activation, respectively, in cells treated with the combined drugs (). Collectively, these data indicated that DMAQ-B1 and AKT inhibitor VIII treatment alter IIS phosphorylation in Ae. aegypti cells in a pattern consistent with the activation of FOXO- and ERK-dependent antiviral signaling independent of viral infection.

Based on these effects on RNAi and JAK/STAT signaling in the absence of virus, we sought to determine the effects of single and combined drugs on ZIKV replication in Ae. aegypti cells. Aag2 cells were primed with individual and combined drugs for 24 h prior to infection with the clinically isolated PRVABC59 strain of ZIKV (). We observed significant reductions in ZIKV titer in cells treated with individual and combined drugs by 3 days post-infection (d p.i.). Most notably, ZIKV titers were undetectable by 3 d p.i. in cells treated with the combined drugs (). Patel and Hardy (2012) showed that AKT inhibitor VIII was antiviral in Sindbis virus (SINV)-infected Aedes albopictus C6/36 cells [28], but the dysfunctional RNAi response in these cells [29] precluded the confirmation of mechanism in its entirety. Accordingly, we concluded that DMAQ-B1 and AKT inhibitor VIII treatments induced an antiviral response that was increased to the point of non-detectable ZIKV titers when these treatments were combined.

DMAQ-B1- and AKT inhibitor VIII-supplemented sucrose water induces activation of RNAi and JAK/STAT signaling in Aedes aegypti

To model established sugar-bait strategies implemented in the field [13] and translatability of our in vitro findings, we next evaluated the effect of DMAQ-B1 and AKT inhibitor VIII treatment against the IIS-dependent antiviral response in adult female Ae. aegypti. Aged-matched 6-9-day old female mosquitoes were continuously exposed to 10% sucrose water supplemented with either 1% DMSO vehicle control, individual, or combined 10 μM DMAQ-B1 and 10 μM AKT inhibitor VIII. Survival was measured over a 14-day treatment period with no difference between treatment conditions (). Whole mosquitoes were collected at 3 (), 7 (), and 11 day () for analysis of RNAi and JAK/STAT gene induction by qRT-PCR. AGO2 and p400 were examined as markers of RNAi () [30,31], while Vago2 and vir-1 were examined as downstream effectors of JAK/STAT () [32,33]. At day 3 we observed higher expression of RNAi-related genes in combined drug-treated mosquitoes compared to our vehicle control () but no difference in JAK/STAT-related genes (). At day 7, however, we saw that RNAi and JAK/STAT gene induction were significantly higher in our combined drug-treated mosquitoes compared to our vehicle control (). By day 11 we observed a loss in immune gene induction on the combined drug-treated mosquitoes (). This leads us to infer that the effectiveness and activity of DMAQ-B1 and AKT inhibitor VIII, at least in combination, induces RNAi and JAK/STAT genes is optimal up to 7 days of continuous treatment but gene induction is reduced by 11 days, possibly due to negative feedback that inhibits the induction of these genes during continuous feeding. Because of this limited toxicity, transcript profile, and range of efficacy, we conclude that DMAQ-B1 and AKT inhibitor VIII induces RNAi and JAK/STAT signaling in a similar manner as we observed in vitro.

Continuous drug treatment via sucrose water results in induction of RNAi and JAK/STAT signaling in Aedes aegypti mosquitoes.

Induction of RNAi and JAK/STAT gene transcripts at (A-D) 3 d, (E-H) 7 d, and (I-L) 11 d treatment was measured in whole mosquitoes receiving continuous treatment of vehicle, 10 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs via sucrose water. Transcripts were measured for (A, E, I) AGO2, (B, F, J) p400, (C, G, K) Vago2, and (D, H, L) vir-1 by qRT-PCR (*p < 0.05; **p<0.01, Unpaired t test with Welch’s correction for multiple comparisons). Closed circles represent individual replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Horizontal bars represent mean and error bars represent SD. Results represent duplicate independent experiments.

Bloodmeal treatment of DMAQ-B1 and AKT inhibitor VIII in ZIKV-infected Aedes aegypti induced simultaneous activation of RNAi and JAK/STAT signaling

Based on IIS-dependent antiviral activity of DMAQ-B1 and AKT inhibitor VIII in vitro and in vivo, we sought to evaluate whether similar drug effects could be detected in Ae. aegypti adult females during ZIKV infection. Aged-matched 6–9-day old female mosquitoes were fed a ZIKV-containing bloodmeal including vehicle, individual, or combined 10 μM DMAQ-B1 and 10 μM AKT inhibitor VIII. Drug concentrations were selected based on mortality studies to measure drug lethality to mosquitoes over a dose range (). Mosquitoes were collected at 3, 7, and 11 d p.i., timepoints that corresponded with complete digestion of the blood meal, progression of viremia into distal tissues, and virus infection of the salivary glands [34-36]. Expression levels of RNAi and JAK/STAT signaling gene products were quantified by qRT-PCR at 7 d p.i. () and 11 d p.i. (). AGO2 and p400 were examined as markers of RNAi () [30,31], while Vago2 and vir-1 were examined as downstream effectors of JAK/STAT () [32,33]. Similar to previously discussed findings in Aag2 cells and non-infected mosquitoes, we observed that the combination drug treatment resulted in higher expression of RNAi and JAK/STAT signaling gene products at 7d p.i. (). Interestingly, at 11 d p.i., only transcript levels for AGO2 remained significantly higher for individual drug- and combination drug-treated mosquitoes (). This gene expression profile is similar to mosquitoes that were continuously treated with individual or dual-drug supplemented sucrose water. Based on high transcript expression at 3 d p.i. in mosquitoes treated with the drug combination (), we demonstrate loss of gene induction between 7 and 11 d p.i. suggesting that drug treatment may have a limited efficacy by 11 d post-bloodmeal regardless if DMAQ-B1 and AKT inhibitor VIII is introduced in a single blood-feed or a multiday exposure. Expression levels of other related RNAi genes including Dicer2 and Ppo8 [30,37] and JAK/STAT dome [38] were enhanced in combined drug-treated mosquitoes 7 d p.i. () which affirms the effect that DMAQ-B1 and AKT inhibitor VIII have for RNAi and JAK/STAT signaling in Ae. aegypti mosquitoes.

Combined drug treatment induced activation of RNAi and JAK/STAT in Aedes aegypti at 7 d p.i. that was reduced by 11 d p.i.

Induction of RNAi and JAK/STAT gene transcripts at (A-D) 7 d p.i. and (E-H) 11 d p.i. was measured in whole mosquitoes infected with ZIKV and treated with vehicle, 10 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs. Transcripts were measured for (A, E) AGO2, (B, F) p400, (C, G) Vago2, and (D, H) vir-1 by qRT-PCR (*p < 0.05; **p<0.01; ***p<0.001; **** p<0.0001, Unpaired t test with Welch’s correction for multiple comparisons). Closed circles represent individual replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Horizontal bars represent mean and error bars represent SD. Results represent duplicate independent experiments.

DMAQ-B1 and AKT inhibitor treatment reduced infection prevalence and ZIKV titer in Aedes aegypti

We next sought to evaluate the effects of individual and combined drug treatments on infection prevalence and ZIKV titers in adult mosquitoes. Mosquitoes were fed a ZIKV-containing bloodmeal treated with vehicle, DMAQ-B1, AKT inhibitor VIII, or combined drug treatment as described. We collected mosquitoes at 3, 7, and 11 d p.i. and analyzed individual midguts, pairs of salivary glands, and carcasses for ZIKV titers (n = 30). There were no differences in virus infection prevalence or viral titers at 3 d p.i. (). At 7 d p.i. infection prevalence was significantly reduced midgut and salivary gland tissues but not carcasses from combined drug-treated mosquitoes (). The titer of ZIKV-positive mosquitoes at 7 d p.i., however, was only reduced in the midguts of combined drug-treated mosquitoes with an approximate 30-fold reduction. There was no difference in carcass and salivary gland titers (). By 11 d p.i., both infection prevalence (), and viral titer () were significantly reduced across tissues in mosquitoes treated with individual and combined drug treatment relative to vehicle control. Titers in ZIKV-positive mosquitoes were reduced by ~42-fold in carcass, ~15-fold in midgut, and ~24-fold in salivary glands (). Notably, infection prevalence and salivary gland viral titers were reduced in combined drug-treated mosquitoes at 11 d p.i., a time consistent with virus transmission during feeding [39,40]. Mosquitoes that received the combined drug treatment were not only less likely to be ZIKV-positive, but salivary gland viral load was also substantially reduced in positive mosquitoes. These observations suggested that combined drug treatment and coordinated activation of RNAi and JAK/STAT provides antiviral immunity against ZIKV that effectively reduced infection prevalence and viral load compared to vehicle controls. These effects of combined drug treatment would be predicted, therefore, to reduce mosquito transmission of ZIKV.

Individual and combined drug treatments reduced infection prevalence and ZIKV titers in Aedes aegypti.

Individual mosquito midguts, pairs of salivary glands, and carcasses (n = 30) were titered for ZIKV by standard plaque assay at (A-F) 7d p.i. and (G-L) 11 d p.i. (A-C, G-I) Infection prevalence was calculated as the ratio of ZIKV-positive samples to the total sample size (*p < 0.05; **p<0.01; ***p<0.001, Two-tailed Fisher’s exact test). (D-F, J-L) Viral titers were determined in ZIKV-positive mosquitoes (*p<0.05; **p<0.01; ***p<0.001; **** p<0.0001, Unpaired t test with Welch’s correction for multiple comparisons). Closed circles represent individual replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Horizontal bars represent mean and error bars represent SD. Results represent duplicate independent experiments.

Inhibition of RNAi and JAK/STAT signaling resulted in loss of drug-mediated antiviral protection

To confirm that DMAQ-B1 and AKT inhibitor VIII mediated-antiviral protection is via RNAi- and JAK/STAT-dependent responses, we transfected Aag2 cells with siRNA constructs to knockdown expression of AGO2 and vir-1. Cells were transfected with siRNAs that targeted either gene individually (siAGO2, siVir-1) or stacked gene expression (siAGO2 + siVir-1) [41]. We measured AGO2 and vir-1 expression at 48, 72, and 120 h in accordance with siRNA removal, when cells were infected, and when supernatant was collected, respectively (). We observed significantly reduced gene expression at 48 h and 72 h post transfection for both individual and stacked siRNA treatments compared to cells that were treated with non-targeting siRNAs as a control [42]. Individual siAGO2 treated cells exhibited a 68% and 41% reductions in AGO2 expression at 48 h and 72 h, respectively, whereas stacked siRNA treated cells exhibited 84% and 99% reduction at the same timepoints (). We observed that AGO2 expression returned to control levels in our siAGO2 and siAGO2 + siVir-1 transfected cells by 120 h. Expression levels of vir-1 in siVir-1- and stacked transfected cells were reduced at all timepoints measured (). In siVir-1-transfected cells, vir-1 expression was reduced by 69% at 48 h, 88% at 72 h, and 74% at 120 h. In stacked-transfected cells, vir-1 expression was reduced by 82% at 48 h, 77% at 72 h, and 63% at 120 h. Finally, we treated cells at 48 h post siRNA transfection with vehicle, individual, or combined drug treatments for 24 h prior to ZIKV infection. Viral titers were measured in supernatants collected at 2 d p.i. to determine if drug-mediated antiviral protection was impacted in the absence of antiviral RNAi, JAK/STAT, or both. We observed that both individual and combined AGO2 and vir-1 knockdowns resulted in significant losses of drug-mediated antiviral protection relative to drug-treated controls (). Interestingly, while we observed a loss in antiviral protection when comparing all data among gene knockdown groups (), we observed a significant increase in viral titers only among the vehicle control groups when comparing siControl to siVir-1 or combined knockdown (). One explanation for why we did not observe differences between the siControl and siAGO2 group during vehicle treatment may be that while AGO2/RNAi is inhibited at the time of infection, its activity is restored by the time of sample collection (). Another possibility is that while IIS-dependent RNAi and JAK/STAT signaling are sufficient to significantly reduce ZIKV titers, other pathways may also contribute to this biology. For example, despite the reduction in ZIKV to undetectable levels via IIS-dependent antiviral immunity, Toll signaling [20] and autophagy [43] could contribute to control of ZIKV replication. Collectively, our data suggest that repurposing small molecule drugs to target mosquito IIS can induce antiviral responses that significantly reduce ZIKV infection prevalence and transmission potential in Ae. aegypti through activation of RNAi and JAK/STAT signaling as inhibition of either or both pathways result in a loss of drug-mediated antiviral protection.

Knockdown of RNAi and JAK/STAT signaling resulted in loss of drug-mediated antiviral protection.

(A) Experimental schematic illustrating process in which cells are transfected with siRNA for 48 h, drug-treated for 24 h prior to infection (72 h), and viral titer measured at 2 d p.i. (120 h). (B-C) AGO2 and vir-1 were knocked down in Aag2 cells and transcript levels were determined for (A) AGO2 and (B) vir-1 by qRT-PCR for cells transfected with scramble control (siControl), individual siRNA construct, or stacked siRNA (siAGO2+siVir-1) at 48 h, 72 h, and 120 h (**p<0.01; ***p<0.001; **** p<0.0001, Unpaired t test with Welch’s correction). (D) 48 h following transfection, cells were primed with DMAQ-B1 or AKT inhibitor VIII for 24 h prior to infection with ZIKV (MOI = 0.01 PFU/cell). Supernatant was collected at 2 d p.i. and virus was titered by standard plaque assay (*p<0.05; **** p<0.0001,Two-way ANOVA with uncorrected Fisher’s LSD test). Closed circles represent individual well replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Results are presented as pooled data of triplicate, independent experiments.

Discussion

Global climate change has enabled the expansion of mosquito populations into new ranges with concomitant increases in the variety and incidence of mosquito-borne diseases. Recent ZIKV epidemics have demonstrated the need for research focused on identifying novel and more effective targets at the vector level. Current vector control efforts involving microbiota or genetic manipulations, while promising, could be enhanced by the addition of antiviral drug strategies to ongoing control efforts.

In the present work, we evaluated the potential of IIS-targeted small molecules to reduce ZIKV infection prevalence and titers in Ae. aegypti. We demonstrated that the potent insulin mimetic DMAQ-B1 and the AKT inhibitor VIII synergized IIS-mediated antiviral immunity in Ae. aegypti to reduce ZIKV infection prevalence and titers in infected mosquitoes (). While this study is not the first to identify IIS regulation of antiviral immunity, we have advanced this field by demonstrating that readily available and potent IIS-targeted small molecules induced substantial and significant antiviral immunity in Ae. aegypti against a clinically virulent strain of ZIKV. By targeting IIS as a mediator of two independent antiviral pathways, we reduced both infection prevalence and virus titers, outcomes predicted to reduce the likelihood of transmission. Both of these effects would be predicted to reduce ZIKV transmission by the primary vector Ae. aegypti. In demonstrating these effects, we have also provided a foundation for future translation of our findings to the field. We have demonstrated that exposure of DMAQ-B1 and AKT inhibitor VIII through either continuous exposure () or a single treatment () was sufficient to induce activation of both antiviral pathways independently of ZIKV infection. More importantly, even with the limited time range in which these drugs appear to induce pathway activation, there was a significant reduction in ZIKV positive- and vector competent-mosquitoes following IIS pathway activation (). Specifically, we seek to advance small molecule delivery via attractive bait stations to induce IIS-mediated, broad antiviral immunity in mosquitoes that ingest these compounds.

Selective targeting of insulin-signaling in Ae. aegypti impacts canonical antiviral responses that can effectively reduce ZIKV replication and likelihood of transmission.

Schematic of proposed mechanism of antiviral action mediated by DMAQ-B1 and AKT inhibitor VIII during ZIKV infection through simultaneous induction of antiviral RNAi and JAK/STAT signaling.

Of particular interest for a potential field-based strategy is the broad impact that IIS appears to have across species. Previous studies have confirmed that exogenous treatment with or the endogenous effects of insulin in D. melanogaster and Culex spp. reduced replication of both WNV and DENV [14,44]. While this broad-antiviral effect is promising, further investigation is necessary to elucidate the full impact our proposed small molecules may have when introduced as a field-based strategy. Because of the limited studies on the effects of DMAQ-B1 and AKT inhibitor VIII on insects, it is unknown if these drugs may be hazardous to beneficial species that could be exposed during mosquito treatment [13,45].

Interestingly, the antiviral effects of IIS during ZIKV infection are not limited to arthropod species. In mammalian models, ZIKV NS4A/NS4B activates PI3K-AKT signaling that is associated with neurogenetic dysregulation [46]. Further, the broadly antiviral celecoxib kinase inhibitor AR-12 and AKT inhibitor VIII have been shown to reduce ZIKV replication and pathogenesis in mice by blocking PI3K-AKT activation [47]. It is also established that diabetic individuals with dysfunctional IIS are more susceptible to severe disease during WNV [48,49], DENV [50], and ZIKV infection [51].

Given the variety of mosquito species that deploy IIS-dependent immunity against notable major arboviruses, it would be worth investigating whether similarly broad IIS regulation of antiviral responses can be detected in mammalian hosts. If so, it may be possible to develop IIS-targeted transmission blocking therapeutic drugs that mitigate Zika disease and, when delivered in blood from treated patients to Ae. aegypti, reduce infection in and transmission by the mosquito vector.

Materials and methods

Mosquito rearing

Aedes aegypti strain Poza Rica, from the state of Veracruz, Mexico were originally collected in 2012, and maintained as previously described [35]. Adult mosquitoes were provided continuous access to water and 10% sucrose ad libitum using soaked cotton balls that were replaced every other day prior to and following bloodmeal feeding. Females were allowed to feed on defibrinated sheep blood (Colorado Serum Company 31123) supplemented with 1mM ATP using an artificial feeding system to stimulate oogenesis. Larvae were reared and maintained under constant 28°C, 70% humidity, and 12-hour light, 12 hour dark diurnal cycle. 6–9 day old adult female mosquitoes were deprived of sucrose 24 hours prior to experimental feedings as described [35]. Mosquito infections, maintenance, and plaque assays were performed under BSL3 and ACL3 facilities, approved by Colorado State University’s Institutional Biosafety Committee 16-074B.

Cells and virus

Vero cells (ATCC, CRL-81) were provided by A. Nicola and cultured at 37°C/5% CO2 in DMEM (ThermoFisher 11965) supplemented with 10% FBS (Atlas Biologicals FS-0500-A) and 1x antibiotic-antimycotic (ThermoFisher 15240062). Ae. aegypti Aag2 cells (Wolbachia-free) [41] were gifted by S. O’Neill and cultured as described in [41]. For drug treatment, culture media with 2% FBS were supplemented with 1% DMSO, 1 μM Demethylasterriquinone B1 (DMAQ-B1) (R&D Systems 1819/5), 10 μM AKT inhibitor VIII (Sigma Aldrich 124018), or combined drugs. Concentrations of DMAQ-B1 and AKT inhibitor VIII were selected at non-cytotoxic levels for both cell culture () and adult mosquitoes (). ZIKV strain PRVABC59 (Accession # KU501215) was obtained from the CDC and was isolated in 2015 from a clinical case in Puerto Rico and prepared as described [35].

In vitro virus replication

Aag2 cells were seeded into a 24-well plate at a confluency of 5×105 cells/well with 6 independent wells for each experimental condition. The following day, cells were treated with 1% DMSO, 1 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs in 2% FBS media as described [14] for 24 h prior to infection. Cells were then infected with ZIKV at MOI of 0.01 PFU/cell for 1 h. Virus inoculum was removed, and fresh experimental media was added. Supernatant samples were collected at 1 and 3 d p.i. for later titration. ZIKV titers were determined by standard plaque assay on Vero cells [52,53].

Cytotoxicity of DMAQ-B1 and AKT inhibitor VIII

Cytotoxicity of DMAQ-B1 and AKT inhibitor VIII was evaluated in both cell culture and in adult female Ae. aegypti. DMAQ-B1 and AKT inhibitor VIII was added to a monolayer of 2.5×105 cells/well in 48-well plates at various concentrations (100 μM, 10 μM, 1 μM, 0.1 μM). Cells were collected at 1, 2, and 3 d post-treatment, stained with trypan blue (ThermoFisher 15250–061) and scored as live or dead as described [54]. Combined DMAQ-B1 and AKT inhibitor VIII cytotoxicity was evaluated using the maximum individual concentrations that corresponded to minimal cytotoxicity. A total of eight technical replicates were averaged for each biological replicate. 1% DMSO treated and 1% Triton X-100 treated cells were also scored as negative and positive controls, respectively. Toxicity was evaluated similarly in 6–9 day old female mosquitoes treated by either drug-treated sucrose water or drug-treated bloodmeal. Sucrose water-treated mosquitoes were provided 10% sucrose water with either 10 μM DMAQ-B1, 10 μM AKT VII inhibitor, combined treatment of both 10 μM DMAQ-B1 and 10 μM AKT inhibitor, or 1% DMSO vehicle control ad libitum. Mosquitoes were maintained for 14 d to monitor mortality. Bloodmeal-treated mosquitoes were fed a bloodmeal containing small molecule drugs (100 μM, 10 μM, 1 μM), 1% DMSO vehicle control, or blood only. Following 1 h of feeding, engorged females were kept and maintained on sucrose for 14 d to monitor mortality. Combined small molecule drug treatment was evaluated using observed lethal and nonlethal individual concentrations. Each experimental group contained approximately 70–100 mosquitoes.

Immunoblotting

Protein extracts were prepared by lysing cells with RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1mM Na3VO4, 1 mM NaF, 0.1 mM PMSF, 10 μM aprotinin, 5 μg/mL leupeptin, 1 μg/mL pepstatin A). Protein samples were diluted using 2x Laemmli loading buffer, mixed, and boiled for 5 minutes at 95°C. Samples were analyzed by SDS/PAGE using a 10% acrylamide gel, followed by transfer onto PVDF membranes (Millipore IPVH00010). Membranes were blocked with 5% BSA (ThermoFisher BP9706) in Tris-buffered saline (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and 0.1% Tween-20 for 1 h at room temperature.

Primary antibody labeling was completed with anti-P-Akt (1:1,000; Cell Signaling 4060), anti-P-ERK (1:1000; Sigma M8159), anti-P-FOXO (1:1000; Millipore 07–695), or anti-actin (1:10,000; Sigma A2066) overnight at 4°C. Secondary antibody labeling was completed using anti-rabbit IgG-HRP conjugate (1:10,000; Promega W401B) or anti-mouse IgG-HRP conjugate (1:10,000; Promega W402B) by incubating membranes for 2 h at room temperature. Blots were imaged onto film using luminol enhancer (ThermoFisher 1862124). Densitometry analysis was completed using three independent blots using BioRad Image Lab and GraphPad Prism 9 with bands normalized to actin.

RNA interference in vitro

Long dsRNA targeting Ae. aegypti AGO2, vir-1, and non-targeting control dsRNA was synthesized as described [41]. Targeted sequences and primers are listed in . dsRNA was transfected into Aag2 cells as described [41] for 48 h prior to small molecule treatment and infection. RNA was extracted and purified to confirm reduced expression by qRT-PCR at 48 h, 72 h, and 120 h post transfection. Viral concentration was confirmed by standard plaque assay at 2 d p.i.

Quantitative reverse transcriptase PCR

qRT-PCR was used to measure mRNA levels in Ae. aegypti Aag2 cells and adult females. Cells or mosquitoes were lysed with Trizol Reagent (ThermoFisher 15596). RNA was isolated by column purification (ZymoResearch R2050), DNA was removed (ThermoFisher 18068), and cDNA was prepared (BioRad 170–8891). Expression of Ae. aegypti AGO2, p400, Vago2, and vir-1 were measured using SYBR Green reagents (ThermoFisher K0222) and normalized to actin to measure endogenous gene levels for all treatment conditions. The reaction for samples included one cycle of denaturation at 95°C for 10 minutes, followed by 45 cycles of denaturation at 95°C for 15 seconds and extension at 60°C for 1 minute, using an Applied Biosystems 7500 Fast Real Time PCR System. ROX was used as an internal control. qRT-PCR primer sequences are listed in .

Immunofluorescence microscopy

Ae. aegypti Aag2 cells were seeded onto coverslips in 12-well plates at a confluency of approximately 1x106 cells/well. Cells were then treated for 24 hours with 1% DMSO, 1 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined small molecule treatment supplemented in 2% FBS media as described [14]. Coverslips were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized in 0.1% Triton-X-100 for 30 minutes at room temperature and blocked in 1% BSA in TBS for 30 min at 37°C. Primary antibody labeling was completed with anti-P-FOXO (1:100) and anti-P-ERK (1:100) for 2 h at humified room temperature. Secondary antibody labeling was completed using anti-rabbit (Life Technologies A11034) or anti-mouse (Life Technologies A11029) Alexafluor 488 (1:300) by incubating membranes for 1 h at room temperature in the dark. Samples were stained with DAPI (1:100; Cell Signaling 4083), mounted onto coverslips using ProLong Diamond Antifade Mountant (Invitrogen P36961), and imaged using a Leica Sp8X confocal microscope. Localization percentages were determined using Adobe Illustrator 2021 by counting the total number of cells and evaluating if green-fluorescent signal was cytosolic, nuclear, or no signal in relation to DAPI-stained nuclei.

Mosquito infections

Fresh ZIKV virus stock was made from Vero cells infected at MOI of 0.1 PFU/cell at 72 hours prior to bloodmeal feed of 6–9 day old female mosquitoes as described [35,36]. Mosquitoes were fed a bloodmeal supplemented with 1 mM ATP and infected with the fresh virus inoculum that was back-titrated to 2.4×106 PFU/mL. Bloodmeals included 1% DMSO vehicle control, 10 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs. Following 1 h of feeding, mosquitoes were anesthetized on ice and engorged mosquitoes were moved into new cartons and maintained on sucrose. Mosquitoes were collected at 3, 7, and 11 d p.i. in which the midgut, salivary glands, and carcass were separated, homogenized, and filtered for infection determination and viral titer. Whole mosquitoes were collected at the same timepoints for qRT-PCR analysis.

Quantification and statistical analysis

Results presented as dot plots show data from individual biological replicates (n = 3–18), the arithmetic mean of the data, shown as a horizontal line. Biological replicates of adult mosquitoes (n = 2–24) consisted of two pooled mosquitoes. Results shown are representative of at least duplicate independent experiments, as indicated in the figure legends. All statistical analyses of biological replicates were completed using GraphPad Prism 9 and significance was defined as p<0.05. One-way ANOVA with Tukey’s correction for multiple comparisons was used for densitometry analysis. Two-way ANOVA with Tukey’s correction for multiple comparisons was used for microscopy localization analysis. Unpaired t test with Welch’s correction was used for qRT-PCR and in vivo viral titer analysis. Two-way ANOVA with Tukey’s correction was used for analysis of multiday in vitro viral titer. Two-tailed Fisher’s exact test was used to compare infection prevalence. Two-way ANOVA with Tukey’s correction for multiple comparisons was used for analysis of small molecule cytotoxicity in vitro and in vivo. All error bars represent standard deviation (SD) of the mean. Outliers were identified using a ROUT test (Q = 5%) and removed.

qRT-PCR Primers.

(DOCX)

Click here for additional data file.

Statistical analysis of data presented in Fig 5D.

(XLSX)

Click here for additional data file.

DMAQ-B1 and AKT inhibitor VIII exhibited dose-dependent cytotoxicity in Aag2 cells.

Aag2 cells were treated with various concentrations of (A) DMAQ-B1, (B) AKT inhibitor VIII, (C) combined drugs, or DMSO vehicle control and cell viability was measured by trypan blue exclusion. Cells that received 1% Trixton-X-100 treatment were used as a positive, 100% lethality control. Closed circles represent biological replicates measured in technical triplicate. Horizontal black bars represent the mean. Error bars represent SD. Significance was measured by Two-Way ANOVA with 1% DMSO vehicle control (*p<0.01). Data are representative of triplicate independent experiments.

(TIF)

Click here for additional data file.

Continuous DMAQ-B1 and AKT inhibitor treatment via sucrose water does not impact mosquito survival.

Adult female Ae. aegypti were given sucrose water supplemented with 1% DMSO (vehicle), 10 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs ad libitum and toxicity was measured by survival over 14 days. Closed circles represent percent survival of mosquitoes (n = 60–100) measured in triplicate. Horizontal black bars represent the mean. Error bars represent SD. Significance was measured by Two-Way ANOVA with 1% DMSO vehicle control. Data are pooled triplicate independent experiments.

(TIF)

Click here for additional data file.

DMAQ-B1 and AKT inhibitor VIII exhibited minimal, dose-dependent toxicity to Ae. aegypti in bloodmeal.

Adult female Ae. aegypti were treated with various concentrations of (A) DMAQ-B1, (B) AKT inhibitor VIII, (C) combined drugs and toxicity was measured by survival over 14 days. Closed circles represent percent survival of mosquitoes (n = 60–100) measured in triplicate. Horizontal black bars represent the mean. Error bars represent SD. Significance was measured by Two-Way ANOVA with 1% DMSO vehicle control (*p<0.05). Data are representative of duplicate independent experiments.

(TIF)

Click here for additional data file.

RNAi and JAK/STAT signaling was induced in small molecule treated mosquitoes at 3 d p.i.

Induction of (A) AGO2, (B) p400, (C) Vago2, and (D) vir-1 in adult female Ae. aegypti was measured by qRT-PCR 3 d p.i. of ZIKV- and drug-containing bloodmeal. (*p<0.05; **p < 0.01; ***p < 0.001). Open circles represent individual biological replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Horizontal black bars represent the mean. Error bars represent SDs. Data are representative of duplicate independent experiments.

(TIF)

Click here for additional data file.

Additional RNAi and JAK/STAT genes were induced in small molecule treated mosquitoes at 3, 7, and 11 d p.i.

Induction of additional immune genes at (A-C) 3, (D-F) 7, and (G-I) 11 d p.i. in adult female Ae. aegypti was measured by qRT-PCR. RNAi associated genes (A, D, G) Dicer2 and (B, E, H) Ppo8 and JAK/STAT (C, F, I) dome were measured. (*p < 0.05; **p<0.01; ***p<0.001; **** p<0.0001, unpaired t test with Welch’s correction for multiple comparisons). Closed circles represent individual replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Horizontal bars represent mean and error bars represent SD. Results represent duplicate independent experiments.

(TIF)

Click here for additional data file.

Infection prevalence and ZIKV titers were not different among small molecule-treated and control Ae aegypti at 3 d p.i.

Ae. aegypti were primed with 1% DMSO, 10 μM DMAQ-B1, 10 μM AKT inhibitor VIII, or combined drugs and infected with ZIKV by bloodmeal. Mosquitoes (n = 30) were collected at 3 d p.i. and individual midguts, pairs of salivary glands, and carcasses were prepared and titered by standard plaque assay. Infection prevalence was determined by comparing the number of mosquitoes with detectable virus to the total mosquitoes in the sample. Viral titer was measured in mosquitoes that were positive for ZIKV. There were no differences in infection prevalence or viral titers among conditions. Open circles represent biological replicates. Outliers were identified using a ROUT test (Q = 5%) and removed. Bars represent the mean. Error bars represent SDs. Data are representative of duplicate independent experiments.

(TIF)

Click here for additional data file.

31 Oct 2021

Dear Dr. Goodman,

Thank you very much for submitting your manuscript "Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

As you will see, both reviewers suggest that additional experiments are necessary for PP and for fulfilling the comprehensive nature of the research as promised by the paper.

Reviewer 1 suggests really only 1 experiment. Reviewer 2 offers numerous options, suggested that several of these may suffice.

The authors will need to tackle some of these experiments for a revision to be considered.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Elizabeth A. McGraw, PhD

Associate Editor

PLOS Pathogens

Sonja Best

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

As you will see, both reviewers suggest that additional experiments are necessary for PP and for fulfilling the comprehensive nature of the research as promised by the paper.

Reviewer 1 suggests really only 1 experiment. Reviewer 2 offers numerous options, suggested that several of these may suffice.

The authors will need to tackle some of these experiments for a revision to be considered.

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In “Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti”, Trammell and colleagues utilized in vitro and in vivo models to advance the understanding of ingested vertebrate factors of the insulin/insulin-like growth factor signaling cascade could simultaneously induce both the JAK/STAT and RNAi antiviral pathways and decrease infection of Aedes aegypti by Zika virus (ZIKV). Given the dearth of vaccines and therapeutics, the only methods for controlling most arbovirus outbreaks are avoidance and abatement of mosquitoes, and use of insecticides. Therefore, there is an enormous value in methods of modulating mosquito infectability by arboviruses.

The work presented in well-conducted, utilizing appropriate controls and thoughtfully designed while the results are quite promising and are a clear advancement to the field. Despite this however, there is some concern in the design of the in vivo element of the experiment, that

Is elaborated on specifically below. In addition, minor grammatical and writing suggestions are noted.

Reviewer #2: The present paper titled ‘Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti’ by Trammell et al., describes the antiviral properties of two small molecules which induce the RNAi and Jak/STAT pathways. The authors use mostly qRT-PCR to detect RNAi and Jak/STAT pathway gene expression to determine the impact of drug treatment on these pathways and virus plaque assay to detect the impact on virus replication. The major conclusions that the authors make are that a combination of two drugs (which feed into insulin signaling) can increase antiviral responses, specifically RNAi and Jak/STAT, and reduce virus transmission in vivo.

The paper is generally well-written and organized. The topic of study should be of interest to readers of PLOS Pathogens, but this reviewer has some concerns with the clarity of the data and whether the conclusions are supported.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: 1. While the in vitro data is largely complete, there is some concern about the design of the mosquito experiment. Specifically, the two small molecules were provided to mosquitoes within the bloodmeal itself alongside the virus. Given that the authors note the real-world application of this mechanism as a field-based strategy, such an experimental paradigm is quite unrealistic. This is because mosquitoes would likely encounter the bait stations/lures impregnated with the compounds either before or after ingesting a virus laden bloodmeal. It would therefore be advantageous to present a more field accurate mosquito experiment alongside the experiment currently depicted in Figure 3.

a. Specifically, exposure to mosquitoes to the compounds via the sugar or water offered prior to exposure to the bloodmeal would strengthen the external applicability of the work.

2. The authors have framed the application of this study to mosquito control via bait stations, however in “Dong S, Dimopoulos G. Antiviral Compounds for Blocking Arboviral Transmission in Mosquitoes. Viruses. 2021 Jan;13(1):108.” it was noted that the attractive toxic sugar bait strategy “also attracted and killed nontargeted beneficial insects such as honeybees and parasitoid wasps”. A previous study “Ahlers LRH, Trammell CE, Carrell GF, Mackinnon S, Torrevillas BK, Chow CY, et al. Insulin Potentiates JAK/STAT Signaling to Broadly Inhibit Flavivirus Replication in Insect Vectors. Cell Rep. 2019 Nov;29(7):1946-1960.e5” demonstrated that the Drosophila melanogaster are also affected by insulin. While the compounds in the present study do not appear toxic at the concentrations tested, it is reasonable that these concentrations would also have consequences to other insects that sugar feed. Is there any evidence that these agents would have pleiotropic effects in non-target species causing changes to longevity, fecundity, etc? While follow-up experiments to address this are not within the purview of this paper, this limitation and gap should not be ignored in the discussion.

Reviewer #2: My major concerns are with the distinction of the two pathways (RNAi and JAK/STAT) and what the data is really supporting (see specific issues below). Some experiments indicate a much more complicated overlap between the results of the two drugs used, and Figure 4C, which would be a crucial experiment to identify the mechanism of these drugs, does not clarify but rather confuse interpretation of results. While I think the antiviral effect of the two drugs is undeniable and important, this paper is aiming at a clear mechanistic level of how these drugs work in the context of virus infection, but it is not delivering on that. As I said, my main worry comes from the questions that Figure 4C brings up (see more specific comments below). In addition, there are other functional outputs for RNAi (such as small RNA sequencing) that could have been considered to support that part of the mechanism.

I am not expecting all of the experiments mentioned below, but a set of these could strengthen this paper on the mechanistic side. I think right now the mechanism is not clear and one or two of these experiments should be considered (whichever experiments provide useful/clarifying data):

- repetition of the experiment(s) in Figure 4C

- verification of knockdown in these samples at the time when virus is measured

- quantification of other RNAi and Jak/STAT genes

- knockdown of other RNAi and JAK/STAT genes and addition to Fig 4C

- mimicking the proposed function of the drug through other experiments to indicate whether the drugs are antiviral through the proposed mechanism (so this is related to Fig 4C, but maybe starting further upstream with knockdown of FOXO, ERK, Akt, MEK, IR, etc and subsequent drug treatment)

- other readouts to test any impact on RNAi, such as small RNA sequencing (but there may be other ways to test this)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: 1. Line 38: The word “memetic” is incorrect. The correct word is “mimetic” which the authors use throughout the manuscript

2. Line 49: The statement “particularly those transmitted by mosquitoes” is unnecessary

3. Line 67: The use of the phrase “virus-free” regions is incorrect.

4. Figure 3: There appears to be mismatch between mosquito populations between prevalence and titers. In all prevalence figures (3A-C, 3G-I), 30 individual mosquitoes were screened for presence of virus. For an example, consider the vehicle condition in 3A and 3D. Figure 3A shows 33% of mosquitoes were infected with ZIKV which corresponds to 10 mosquitoes. The corresponding dot plot in D has 9 dots. This appears to be a common occurrence throughout Fig 3. Can this be clarified?

5. Lines 237-239: The phrase “…the need for host-virus interactions research” reads awkwardly. Consider rephrasing

6. Line 242: The word “therapeutic” in entirely inappropriate here

7. Line 277-278: Clarify means by which mosquitoes were given access to sugar and water

Reviewer #2: Figure 4C is highly confusing – not in its design, but the results are confusing. While results for the drug treatments look as expected in the control siRNA samples, it is highly surprising that essentially all other siRNA/drug combinations look identical in ZIKV titers. This result removes the clear explanations for how RNAi and JAK/STAT may be induced by the different drugs. For example, in Figure 1I, the authors show no significant impact of DMAQ-B1 treatment on vir-1 expression, but in Figure 4C, vir-1 silencing removed any effect that DMAQ-B1 had on ZIKV titers – so the effect was dependent on vir-1? And if the impact is so clearly vir-1 dependent, how can it be just as clearly Ago-2 dependent? Essentially, there should be more defined differences between the drug/siRNA combinations to support the model shown in Figure 4D. This figure legend also provides no information on independent replicates or even experimental replicates and this reviewer is a bit concerned about the validity of these results. Maybe some independent replicate experiments are needed to try to understand what is going on? Expression of vir-1 and Ago-2 in these exact samples at the time when virus was harvested also would have been useful (as opposed to just at 48h post transfection).

Another larger concern is the difference in this experimental design to the use of sugar-bait feeding. While I understand that this is an initial study, the authors imply that these small molecules could be sugar-fed to mosquitoes and reduce transmission – sugar feeding can change how/when/how efficiently these drugs are taken up and it would have been nice to see an experiment where mosquitoes are provided a virus containing blood-meal while being held on sugar-water (or other sugar bait) that contains the drugs – the potential constant low-level exposure with targeting to the crop instead of the midgut could impact results in either direction.

Line 193: For Figure 3D-F, the authors state that viral titers were reduced less than 2-fold, however based on the y-axis, the data is shown in a logarithmic scale and was reduced by more than 10-fold in the midguts. Carcass data was unclear/not significant, and the salivary gland data probably had too few data points. I think it might be useful to not condense these descriptions into one sentence, but clearly describe each figure panel. For 11dpi, the authors also just say that it is more than two-fold when it is really more than 10-fold (unless I am misinterpreting something).

Line 198-200: The authors compare their data to another study in which 4.8log10 PFU/mL was determined as the required titer in salivary glands to transmit ZIKV. I think the authors may need to be careful with these numbers and the subsequent sentence about ‘reducing numbers below the level of transmission’ – the reason is that all other salivary glands from control mosquitoes also had titers below this 4.8log10 PFU/mL threshold, indicating that either the methods were not as sensitive here as in the other study, or that none of these mosquitoes were transmission competent (in which case there was no reduction below the threshold since none of the mosquitoes passed it). It does not take away from these impressive results, but the wording should be adjusted a bit in my opinion.

The discussion uses and mentions many of my points above as well and would need some adjustment according to my concerns (e.g. the reduction below transmission potential in line 251, or the proposed sugar-bait feeding that was not addressed here experimentally).

Normalizing the qRT-PCR data to the control would help a bit with interpretation, especially in Figure 4A, were the knockdown of gene expression is shown. By normalizing these (ΔΔct or other fold normalization), interpretation would be more intuitive.

Figure 1H-I: The label on the x-axis says 1 day post infection, but the text and the figure legend do not indicate that these cells were infected. Either the next/legend neglected to say that ago2 and vir-1 levels were measured in infected cells, or the axis should be labeled something like ‘post-treatment’ or similar.

The pathway overview figure (Figure 4D) was very helpful – I am glad it was included, and I understand why it was the last figure of the paper, but I actually looked for something like this right away when starting to read the results, mostly in order to interpret the Western Blot results and how they matched the expectations. But I think it helped me then follow the other results (esp. Figure 1) better too. Maybe it could be introduced early on somehow? Or an adjusted version of it maybe? Just a thought because it would help understand the role of the inhibitors quickly (more so than the text). Figure 4D could be a panel in figure 1 of the paper and then the final figure could somehow link it back to the bigger organismal level - the strategy of mosquito feeding on a treated sugar bait, upregulation of RNAi and Jak/STAT, and reduced ZIKV transmission (?), assuming these mechanisms can be clarified a bit more.

Line 100: reference 40 is also relevant here as one of the first to implicate JAK/STAT in ZIKV antiviral responses – none of the cited references 17-19 seem to be from mosquito-based studies (?).

(Also, Figure 3G-I should be moved up since it these panels are mentioned first in the text.)

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at .

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

23 Feb 2022

Submitted filename: PlosPath_Rebuttal-Letter_Final.docx

Click here for additional data file.

1 Mar 2022

Dear Dr. Goodman,

We are pleased to inform you that your manuscript 'Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Elizabeth A. McGraw, PhD

Associate Editor

PLOS Pathogens

Sonja Best

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):

30 Mar 2022

Dear Dr. Goodman,

We are delighted to inform you that your manuscript, "Coupled small molecules target RNA interference and JAK/STAT signaling to reduce Zika virus infection in Aedes aegypti," has been formally accepted for publication in PLOS Pathogens.

We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Pearls, Reviews, Opinions, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064