A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.


Introduction
Lines 71-72.There are a number of factors in mosquito saliva that have been identified as able of modulating host susceptibility to arbovirus infection, as listed later in discussion.I would suggest rephrasing this here to make this clear.
Lines 86-88.Onset of oedema is very rapid and the role that leukocyte influx has on this process is still to be defined.It is like that both neutrophil influx and oedema occur at the same time based on the published studies cited.

Table S1
Line 123 " We next assessed the role of these salivary proteins in triggering neutrophil influx into the host skin.Thus, 100 ng of purified salivary proteins were individually inoculated into a mouse footpad in a subcutaneous manner".It is not clear why 100ng was chosen and whether this reflects the mass inoculated by a probing mosquito.The amount salivated by mosquitoes, e.g.following forced salivation into oil or media should be define.
Figure 1C -a Students T test has been used incorrectly and doesn't account for multiple testing.One would expect a p value of less than 0.05 for every 20 proteins studied, based on chance differences.Since there are more than 20 groups, this is quite likely.A one-way ANOVA or Kruskal Wallis, depending on whether data is normally distributed, should be applied instead.This also applies to most of the statistics used elsewhere, in which a Students T test has been used.All should be corrected.
Figure 1D-L.It is not clear how leukocyte influx, elicited by injection with AaNRP, compares to mosquito biting and/or injection with equivalent amount of mosquito saliva injection.The authors should offer this comparison for key cell types identified.
Figure 1E,F and L. The authors have only assessed monocyte, macrophage and DC influx at 24 hours post injection.This quite a late timepoint, considering enhancement of arbovirus infection by saliva is evident before 6 hours (including in the studies cited in this manuscript e.g.Styer et al).In addition, previously reported infection of myeloid cells occurs within this time frame, long before the 24-hour time point.Virus life cycle is 8 to 10 hours, which means by 24 hours there would have been several rounds of infection and e.g.dissemination of virus to blood.The authors should expand these analyses to include earlier time points.This is key, as entry of virus permissive cells at 24 hours and later may not have significant bearing on defining the outcome to infection, as virus tends to disseminate from skin to blood / systemically within this time frame.This is also the case for later figures e.g. Figure 5, which only shows 24-hour post infection.
Volume of recombinant protein injected was 20ul, this is many orders of magnitude higher than what a mosquito would deposit.This limitation should be discussed.
Figure 1 and 5. Absence of virus vs. uninfected skin comparison in leukocyte entry kinetics experiments.The purpose of this research is to inform on leukocyte migration during transmission of virus from mosquito to vertebrate skin.Virus is an important activator of chemokines and leukocyte influx, and yet is excluded from figure 1, and the comparator to resting skin missing in figure 5.This should be included.
Figure 2. Validation of macrophage depletion.Skin macrophages were depleted through use of an antibody to F4/80.Such depletions are difficult to get working at high efficiency for non-circulating tissue resident cells such as these.The complete absence of macrophages in the F4/80 Ab treated group seems surprising, however the antibody used to validate macrophage depletion was the same as that used for depletion.This is not suitable as it could simply indicate epitope masking by antibody rather than true cell depletion.Observed alterations in neutrophil recruitment could instead reflect consequences of antibody infusion associated with this specific Ab clone.Findings should reassessed or macrophage depletion validated through alternative means.Furthermore, is F4/80 a good marker for skin macrophages?As they will also deplete other cells in the skin e.g.Langerhans cells.Some macrophage population, e.g.those CD11b+ macrophages derived from bone marrow progenitors are typically low for F4/80.This last point should be discussed e.g.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045180/ Figure 3 and 4 are generally well done, although would be more meaningful if LPS contamination of AaNRP preparation was ruled out.Confirmation of TLR4-MyD88 signalling pathway activation in primary cultures of Macrophages is required, as immortalised RAW cells often generate data that is hard to replicate in terminally differentiated (and thereby relevant) cells.The specificity of the new anti-AaNRP antibody needs to be defined for data in Fig 4B/C to meaningful.As for most of the paper, the wrong statistical test has been used; ANOVA is the correct test if there are more than two groups.
Figure 5 Representative gating of ZIKV positive cells should be shown.Infected cells do not necessarily lead to release of new infectious virus and could also represented phagocytised viral debris.The ability of skin isolated infected leukocytes to release new infectious virus should be defined.Figure 6.The authors need to demonstrate that siRNA KO of AaNRP does not have any unintentional effects on mosquito feeding, and thereby transmission, that is unrelated to their mechanisms.For example, they show ZIKV levels are similar in whole salivary gland homogenates, but the PFU/ul of ZIKV in saliva needs to be assessed (e.g. through forced salivation).Probing time and ability to engorge with blood should also be assessed, as differences in probing time and feeding efficiency could impact on volume of saliva deposited and thereby virus titre.
Figure 7.It is not clear how specific resveratrol is on innate immune responses.Ability of this agent to suppress other TLR4 activating ligands should ideally be defined as a positive control.Resveratrol could be modulating numerous innate immune pathways, and/or myeloid cell frequency in resting skin/blood, independent of AaNRP function.What effect does resveratrol have on anti-viral innate immune pathways e.g.type I IFN and IFN stimulated gene expression?All of these pathways could impact on overall host susceptibility to infection.
Discussion.The results presented here suggest that AaNRP accounts for almost all virus enhancing ability of mosquito saliva.What does this mean for the role of other mosquito salivary proteins, including the finding that AaVA-1 (Sun et al) was pivotal for this process, previously published by this group?Furthermore, the discussion as written here suggests that chemokine expression is the limiting step required for salivaenhancement of virus infection.Some other reports have suggested chemokine expression alone is not sufficient to mediate saliva-induced enhancement of arbovirus infection (e.g Lefteri 2022 that is widely cited here).Chemokine expression is clearly key (as shown here) but is one of a number of steps, and this should be placed into context in the discussion.
Referee #2: SUMMARY In this manuscript, Wang et al. report the identification of a mosquito salivary protein (AaNRP) that triggers the infiltration of neutrophils into the skin.This is important as several prior studies indicated that an influx of neutrophils into the skin enhances arbovirus infection and dissemination in the vertebrate host.The authors further show that this protein triggers the production of chemokines in skin macrophages via activation of MyD88 signaling.Remarkably, the authors identify interactions between AaNRP and murine/human TLR1/TLR4 and show that disrupting these interactions abrogates the induction of chemokines.A series of well-designed studies in mice provide additional evidence that AaNRP enhances ZIKV infection through activation of the MyD88 pathway, promotes establishment and dissemination of ZIKV infection after transmission from infected mosquitoes that express AaNRP in the salivary gland, while KD of AaNRP in mosquitoes abrogates these effects, and that limiting the inflammatory response induced by AaNRP alone, co-delivered with ZIKV, or delivered by mosquito bite can diminish effects and improve outcomes.Overall, this is a comprehensive and careful study that uses a variety of approaches that identifies how AaNRP enhances ZIKV infection.The study should be of substantial interest.A few comments to improve the study are outlined below.

MAJOR 1. Fig 1C:
This experiment could include additional controls beyond PBS.For example, does heat-treatment of the protein sample prior to injection in mice negate the neutrophil influx? 2. Fig 1F -1H.The 1A8 antibody targets both Ly-6C and Ly-6G and thus depletes multiple cells type including monocytes.This experiment is not sufficient to conclude that an influx of neutrophils is a prerequisite for recruitment of other myeloid cells.The authors should perform more specific cell depletion studies to support this conclusion and evaluate additional cell populations to demonstrate the specificity of the depletion.4G-H.These findings suggest that TLR4 is an important recognition receptor for AaNRP.Showing that AaNRP-mediated induction of chemokines is resistant to inhibition by polymyxin B would further support the authors conclusions, as the presence of LPS is a concern.5C could be more informative if the percentage and total number of infected cells within the specific cell type evaluated was reported.That is, among macrophages, did the frequency and total number of infected macrophages increase with AaNRP?Same question for other cell types.

For Fig 5D and other measurements of viremia
, the authors should also assess the amount of infectious virus present, as the approach used could be influenced by differences in circulating cells between the experimental groups.In addition, infectious virus in the blood is more informative.Similarly, due to the changes in cell composition in tissues (e.g., shown in Fig 5B and 5C), using the ZIKV/GAPDH mRNA ratio to quantify viral tissue burden in murine tissues throughout this study could be misleading.MINOR 1. Line 323-324.The authors indicate that experiments were performed to assess flavivirus transmission.This is not an accurate description of the experiments displayed in Fig 5 .ADDITIONAL SUGGESTIONS 1.The authors could consider the use of TLR1 and/or TLR4 deficient animals (which can be treated with an IFNAR1 blocking Ab) to confirm a role for these receptors in AaNRP driven inflammation and enhancement of ZIKV infection in vivo.This would enhance the study and support the author's model regarding mechanism of action.

Referee #3:
This manuscript by Wang and colleagues reports an intriguing investigation into Aedes aegypti salivary proteins that augment ZIKV and DENV infection.The authors reasoned from previous findings that neutrophil recruitment into the skin increases the likelihood of infection establishment and dissemination in the human host.They thus systematically screened recombinant salivary proteins of Ae aegypti to determine candidates that recruit neutrophils into the skin and identified AaNRP.They showed that AaNRP bound to TLR-1 and -4 stimulation and activated canonical MyD88-NFkB signaling to induce expression of chemokines that recruited neutrophils to the site of the mosquito bite.They then showed that inhibition of MyD88 signaling in mice, and knockdown of AaNRP expression in infected mosquitoes both reduced ZIKV and DENV-2 infection in mouse footpad.Similarly, both interventions reduced viremia levels at almost all time points measured.They finally showed that daily prophylactic supplementation with resveratrol, which is known to inhibit MyD88-NFkB signaling, could also reduce ZIKV viremia levels.They concluded that their findings provide mechanistic insights into the augmentation of flaviviral infection by mosquito a salivary protein and that this finding could be used prophylactically to prevent disease.
The topic of this study is interesting and important, given the paucity of information on this subject.The authors have also done a huge amount of work for this manuscript.There are, however, several areas of concern that need additional attention.These are: 1. Line 125 and Figure 1C.The authors screened the panel of proteins for neutrophil recruitment activity by inoculating these proteins subcutaneously into mouse footpads.However, the mosquito proboscis only probes the dermis in humans and cannot extend into the subcutaneous layer.Could there be differences in how antigen presenting cells resident in the dermis or even keratinocytes respond to these proteins and hence influence downstream neutrophil recruitment?The experimental approach could thus have underestimated the number of salivary proteins that could play a role in augmenting ZIKV and DENV infection.
2. What is the normal function of AaNRP?Obviously, neutrophil recruitment is not required for successful completion of bloodmeal for mosquitoes to lay their eggs.
3. For many of the experiments, AaNRP was compared against PBS as a negative control.PBS is not the ideal control.A more appropriate negative control would be another protein, preferably a protein that is also excreted in Ae aegypti saliva.Given the many negative findings in Figure 1C, a negative control could have been chosen from that list of proteins.Such a control would have supported the specificity of AaNRP in mediating the effects shown in the rest of the manuscript.
4. Lines 138-140 and Fig S1G .Increased expression of AaNRP after a bloodmeal does not support the notion proposed in this study, that AaNRP is necessary to augment infection.AaNRP should be measured before and not after a bloodmeal.This could be done by quantifying AaNRP expression from when the mosquito hatches to when it is ready to become gravid through a bloodmeal.
5. Lines 200-201.The approach of using scRNAseq to identify mediators of neutrophil recruitment completely misses a major contributor of acute inflammation -pre-formed mast cell mediators.These pre-formed chemicals are stored in granules that, upon mast cell activation, would be released through degranulation, several of which are known to play roles in neutrophil recruitment.Post-mosquito bite scRNAseq would thus miss many factors that facilitate neutrophil recruitment.
6. Lines 255-256, Figures 2E and F. The data in Figure 2E is incongruent with 2F, the latter showing that anti-F4/80 mAb treatment inhibited neutrophil infiltration compared to isotype IgG control treatment but not to baseline resting levels.As Figure 2E only showed one point per animal, it would be important to know how the data was obtained.How many sections were examined with IHC per animal?How many fields on each slide were viewed and hence sampled to calculate % neutrophil infiltration.Without this information, Figure 2E could be due to biased sampling.

Line 230 and 235.
There is no evidence that AaNRP is "the primary" salivary protein involved in neutrophil recruitment.Neither is there any evidence that it was the "major" factor in upregulation of neutrophil chemoattractants.The authors did not assess the other salivary proteins beyond the first screen which was done through subcutaneous and not intradermal inoculation.Please rephrase.
8. Lines 237-240.Does silencing of AaNRP have off-target effects on mosquito salivary production/secretion? 9. Section under lines 320-321 and Figure 5. Inclusion of a control constituting T6167923 treatment followed by ZIKV infection without AsNRP would have been helpful.Blocking MyD88 signaling could impact ZIKV and DENV infection and thus confound the data reported in this section.
10. Lines 353-354 and Figure 6C.RT-qPCR alone to measure viral load is not sufficient.There is no information on how AaNRP silencing would affect ZIKV lifecycle and maturation.Besides measuring viral RNA, the amount of infectious ZIKV particle in the mosquito saliva should also be shown.
11. Lines 437-456.Resveratrol has pleiotropic effects, some of which, such as increased risk of DNA damage, are harmful.For this compound to be used prophylactically to reduce the level of viremia and hence risk of severe disease, persons living in endemic countries will have to take this drug over long periods.How would the prophylactic benefits weigh against the risk associated with long-term usage of this compound?A more balanced discussion would be helpful.

Dear Editors,
Thank you for the comments on our manuscript (EMBOJ-2023-114972) titled "A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission".We have taken all of the comments into consideration and have revised our manuscript accordingly.We believe that our additional experiments, revised analyses, and modifications of the text have substantially improved the manuscript.
We were encouraged by the positive comments from the editor and reviewers, such as "As you will see from the reports, all reviewers find the study of interest" (Editor), "Overall, this study is nicely done and provides some important new insights…...The role that mosquito saliva has in modulating host susceptibility to virus infection is poorly defined and yet represents a key stage of infection that is common to all viruses transmitted by mosquito to the vertebrate host.As such the findings, if further validated with corrections listed below, are important, novel and of relevance to several key research areas (e.g. virology, immunology, vector biology)."(Reviewer #1), "Overall, this is a comprehensive and careful study that uses a variety of approaches that identifies how AaNRP enhances ZIKV infection.The study should be of substantial interest."(Reviewer #2), "The topic of this study is interesting and important, given the paucity of information on this subject.The authors have also done a huge amount of work for this manuscript."(Reviewer #3).The reviewers also provided important suggestions to improve the manuscript.We have included new experimental data in our manuscript per the reviewers' suggestions.Along with this letter, we provide point-by-point responses to the queries raised by the reviewers.

Responses to Editor's Comments:
Answer: Thank you for these comments.We have taken all these concerns into consideration and have fully solved these concerns by performing enough experiments.We believe solving these concerns has significantly improved our manuscript.The detailed response to each concern is listed below: 1) Exclusion of LPS contamination.Please see #1A Answer.
2) Further support for the role of neutrophils in myeloid cell recruitment.Please see #1G Answer, #1J Answer, #2B Answer and #3E Answer.
3) Assessment of potential effects on mosquito feeding and saliva deposition.Please Responses to Referee #1: #1A.It is common for recombinant proteins made in-house to have LPS contamination, even for those made using insect cell lines.The accidental presence of LPS could activate these responses observed, both in vivo (e.g.neutrophil recruitment) and in vitro assays (e.g.MyD88 pathway activation).TLR2/4 ligands, when co-inoculated with arbovirus are known to enhance infection with arbovirus.It is essential that AaNRP preps are tested for their ability to activate LPS specific responses and / or LPS concentration defined.As the proposed mechanism of action is via MyD88, a kit that that doesn't use this system to detect endotoxin should be employed e.g. the Pierce LAL Chromogenic Endotoxin Quantitation Kit.Even if very low levels of endotoxin are detected, it would then be necessary to show that endotoxin, when spiked into a control recombinant product (e.g. a non-inflammatory mosquito salivary protein other than AaNRP, or scrambled protein) at a similar concentration, does not replicate results observed with AaNRP.#1A Answer: We recognize the reviewer's concern.In this study, the results indicated that AaNRP activates MyD88-dependent canonical NF-κB signaling, which drives the expression of neutrophil chemoattractants and the subsequent recruitment of myeloid cells.The recombinant AaNRP protein was expressed and purified in Drosophila S2 cells.To address the reviewer's concern, we assessed the LPS contamination in the preparation of 1 μg/μl AaNRP and the PBS solution (Gibco, Cat# 10010023) used to dilute AaNRP preps by the Pierce™ Chromogenic Endotoxin Quant Kit (Cat# A39552S).The endotoxin concentration in the preparation of purified AaNRP protein showed no difference with the PBS solution (average level: AaNRP 0.038 EU/ml, PBS 0.037 EU/ml, p=0.66) (Appendix Fig S2A).It is generally believed that an endotoxin content below 0.1 EU/ml has no detectable effect on the cultured cells or animals.In this case, the observed effects should be attributed to AaNRP rather than LPS contamination.To further address the concern of LPS contamination, we next used polymyxin B, a reagent effectively neutralizing LPS, to assess the possibility of LPS-mediated activation of the MyD88-dependent NF-κB pathway.Purified AaNRP and LPS were incubated with sterile water (solvent control) or with 30 μg/ml polymyxin B (neutralization) at 4°C for 24 hours.Then, either sterile water or polymyxin B-treated AaNRP and LPS were added to murine RAW264.7 macrophages for incubation, respectively.Four hours later, the cells were collected for qPCR detection of CXCL1/2/3 expression.The results showed that both sterile water-treated AaNRP and LPS significantly induced the expression of CXCL1/2/3 (Appendix Fig S2B).Polymyxin B preincubation had no influence on AaNRP-induced expression of CXCL1/2/3 but completely abolished LPS-induced CXCL1/2/3 expression (Appendix Fig S2B).These data further validated the specific induction of these chemokines by AaNRP and well addressed the concern of LPS contamination in AaNRP preparation.We have added the data to the revised manuscript (Line 278-293, Page 10 and 11).
Moreover, we included PBS and a heat-inactivated AaNRP (hiAaNRP) as negative controls, and a noninflammatory control (NIC) salivary protein encoded by AAEL009524 as an unrelated control.Thus, 100 ng of each protein or an equal volume of PBS was injected into mouse footpads.The injected skin was later collected for a flow cytometry analysis.Skin injected with PBS, hiAaNRP or the AAEL009524-encoded protein showed similar levels of neutrophils ( Fig #1C.Lines 86-88.Onset of oedema is very rapid and the role that leukocyte influx has on this process is still to be defined.It is like that both neutrophil influx and oedema occur at the same time based on the published studies cited.#1C Answer: Thank you for the reviewer's suggestion.We have revised the sentence to "Thus, neutrophil-driven inflammation leads to an increase in myeloid cells at the mosquito bite site, and these myeloid cells serve as new cellular targets to retain viruses at the inoculation site" (Line 89-92, Page 4).306), which equates to 250-420 ng of salivary proteins.Given the relatively high prevalence of selected components in mosquito saliva (Appendix Table S1), we decided to inoculate 100 ng of a single mosquito saliva protein into one mouse footpad for our investigation.

#1D.
According to the reviewer's suggestion, we further measured the amount of salivary proteins secreted by a mosquito bite under forced salivation into microscopic immersion oil.The amount of salivary proteins secreted by a mosquito bite is approximately 300 ng (Appendix Fig S4C).

#1E. Figure 1C -a Students T test has been used incorrectly and doesn't account for multiple testing.
One would expect a p value of less than 0.05 for every 20 proteins studied, based on chance differences.Since there are more than 20 groups, this is quite likely.A one-way ANOVA or Kruskal Wallis, depending on whether data is normally distributed, should be applied instead.This also applies to most of the statistics used elsewhere, in which a Students T test has been used.All should be corrected.#1E Answer: We recognize the reviewer's concern.We have reanalyzed all the data throughout our study by using appropriate statistical models.The detailed information about statistical analysis has been shown in figure legends and Materials and Methods section of the revised manuscript (Line 934-943, Page 34).

#1F. Figure 1D-L. It is not clear how leukocyte influx, elicited by injection with AaNRP, compares to mosquito biting and/or injection with equivalent amount of mosquito saliva injection. The authors should offer this comparison for key cell types identified.
#1F Answer: Thank you for the reviewer's suggestion.We have accordingly included the control inoculation with an equivalent amount of mosquito saliva.In terms of temporal kinetics, both AaNRP and mosquito saliva were able to activate neutrophil infiltration, which occurred as early as 4 hpi and through 24 hpi with a peak at 12 hpi (Fig 1D).Thus, we assessed the recruitment of myeloid cells toward the inoculated footpads.Notably, monocyte-lineage myeloid cells, such as monocytes, macrophages, and immature dendritic cells (DCs), were highly aggregated at the sites inoculated with either AaNRP or mosquito saliva at 6 hpi (Fig 1E).
In the experimental design in Fig 1F -1H, we would ask if AaNRP-mediated myeloid infiltration was dependent on neutrophils.Indeed, accumulating evidence indicates that inoculation of mosquito saliva results in an influx of inflammatory neutrophils, thus coordinating a localized innate immune response to recruit numerous virus-permissive myeloid cells toward bite sites (Hastings et al., 2019, Journal of virology, 93;Pingen et al., 2016, Immunity, 44: 1455-1469).Subsequently, we assessed the role of AaNRP in neutrophil infiltration and subsequent recruitment of myeloid cells in a mosquito biting model.In this experimental model, the mosquitoes with or without AaNRP silencing in saliva were allowed to bite the footpads of mice.The bitten skin was collected for assessment of neutrophil infiltration and recruitment of monocyte lineages (Fig 1I -L).We believe that the logical designs of these experiments have addressed the reviewer's concern.

#1G. Figure 1E, F and L. The authors have only assessed monocyte, macrophage
and DC influx at 24 hours post injection.This quite a late timepoint, considering enhancement of arbovirus infection by saliva is evident before 6 hours (including in the studies cited in this manuscript e.g.Styer et al).In addition, previously reported infection of myeloid cells occurs within this time frame, long before the 24-hour time point.Virus life cycle is 8 to 10 hours, which means by 24 hours there would have been several rounds of infection and e.g.dissemination of virus to blood.The authors should expand these analyses to include earlier time points.This is key, as entry of virus permissive cells at 24 hours and later may not have significant bearing on defining the outcome to infection, as virus tends to disseminate from skin to blood / systemically within this time frame.This is also the case for later figures e.g.#1H.Volume of recombinant protein injected was 20ul, this is many orders of magnitude higher than what a mosquito would deposit.This limitation should be discussed.#1H Answer: Thank you for the reviewer's suggestion.The saliva volume expectorated by a mosquito is hard to quantify due to very small amounts and individual differences.By using a microtube forced salivation assay, Sanchez-Vargas et al. reported that a single Aedes mosquito salivates an average of 6.82 ± 2.88 nL saliva (Sanchez-Vargas et al., 2019, Insects, 10).Our injected volume is much greater than this natural volume, which is a limitation of our study.We have discussed this limitation in the revised manuscript (Line 541-548, Page 20). 5.This should be included.#1I Answer: As mentioned by the reviewer, both salivary proteins and viruses play critical roles in stimulating the cutaneous influx of chemokines and leukocytes.In this study, the leading aim is to identify the key factor(s) that cause an influx of neutrophils and subsequent recruitment of myeloid cells toward mosquito bite sites.Thus, we did not involve viral factors in the identification of salivary protein(s) triggering the influx of cutaneous neutrophils during mosquito biting ( #1J.Skin macrophages were depleted through use of an antibody to F4/80.Such depletions are difficult to get working at high efficiency for non-circulating tissue resident cells such as these.The complete absence of macrophages in the F4/80 Ab treated group seems surprising, however the antibody used to validate macrophage depletion was the same as that used for depletion.This is not suitable as it could simply indicate epitope masking by antibody rather than true cell depletion.Observed alterations in neutrophil recruitment could instead reflect consequences of antibody infusion associated with this specific Ab clone.Findings should be re-assessed or macrophage depletion validated through alternative means.Furthermore, is F4/80 a good marker for skin macrophages?As they will also deplete other cells in the skin e.g.Langerhans cells.Some macrophage population, e.g.those CD11b+ macrophages derived from bone marrow progenitors are typically low for F4/80.This last point should be discussed e.g.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045180/#1J Answer: We recognize the reviewer's concern.Indeed, the mAb F4/80 (BioxCell, Catalog #BE0206) has been widely used to deplete skin macrophages as well as other noncirculating tissue-resident macrophages.The F4/80-based protocol for the removal of skin macrophages has been generally acknowledged for actual effectiveness We recognize the reviewer's concern for off-target depletion of skin Langerhans cells by inoculation with the F4/80 mAb.Although F4/80 is a good surface marker for dermal and hypodermal macrophages, it is also expressed by some Langerhans cells In addition, the scRNA-Seq data showed that neutrophil chemoattractants such as CXCL1/2/3 were not induced in Langerhans cells after a mosquito bite (Appendix Fig S1).Thus, these results suggest that Langerhans cells may not be important in AaNRP-mediated neutrophil influx, even if they might be nonspecifically targeted by the F4/80 mAb.We have added this discussion to the revised manuscript (Line 549-559, Page 20 and 21).

#1I. Figure 1 and 5. Absence of virus vs. uninfected skin comparison in leukocyte entry kinetics experiments. The purpose of this research is to inform on leukocyte migration during transmission of virus from mosquito to vertebrate skin. Virus is an important activator of chemokines and leukocyte influx, and yet is excluded from figure 1, and the comparator to resting skin missing in figure
We recognize the reviewer's concern about some macrophage populations, e.g., those CD11b+ macrophages derived from bone marrow progenitors are typically low for F4/80.Nonetheless, accumulating evidence indicates that skin resident macrophages are derived from yolk sac progenitors and highly express F4/80 #1K Answer: Thank you for the comments.We have addressed the concerns about the potential LPS contamination of the AaNRP preparation in Answer #1A.According to the reviewer's suggestion, we have reproduced the activation of MyD88-NF-κB signaling by AaNRP in primary murine peritoneal macrophages.AaNRP strengthened the phosphorylation and degradation of IκB-α while activating p65 phosphorylation (Fig 3E,ii).A small molecule MyD88 signaling inhibitor, T6167923, fully abolished the AaNRP-mediated activation of NF-κB signaling in primary murine macrophages, as assessed by the immunoblotting assay (Fig 3E,ii).Furthermore, in the in vivo condition, AaNRP significantly activated the canonical NF-κB signaling axis in mouse footpad skin (Fig EV3G).Overall, we concluded that AaNRP was able to activate MyD88-NF-κB signaling in primary murine macrophages.We have added the results to the revised manuscript (Line 312-314, Page 12).and AaNRP to release progeny infectious viruses at 6 hours post in vitro culture by focus forming assay.First, we isolated CD11b + leukocytes and CD11b -nonleukocytes from the footpad skin coinoculated with ZIKV and AaNRP at 16 hpi by using flow cytometry.CD11b + leukocytes and CD11b -nonleukocytes were cultured in vitro in RPMI 1640 medium with 2% FBS at 37 °C.Six hours later, the supernatants were collected, and the viral titers in the supernatants were determined by focus forming assay.The results showed that the virus-infected leukocytes were definitely able to produce infectious progenies, and in terms of an equal number of cells (5*10^3), CD11b + leukocytes produced many magnitudes of infectious viruses than CD11bnonleukocytes (Fig 5D).This result further corroborated the pivotal role of infiltrated myeloid cells in mediating flavivirus infection.#1O. Figure 6-The authors need to demonstrate that siRNA KO of AaNRP does not have any unintentional effects on mosquito feeding, and thereby transmission, that is unrelated to their mechanisms.For example, they show ZIKV levels are similar in whole salivary gland homogenates, but the PFU/ul of ZIKV in saliva needs to be assessed (e.g. through forced salivation).Probing time and ability to engorge with blood should also be assessed, as differences in probing time and feeding efficiency could impact on volume of saliva deposited and thereby virus titre.#1O Answer: We recognize the reviewer's concern.We therefore assessed whether dsRNA-mediated silencing of AaNRP might cause any unintentional effects on mosquito feeding.In this experiment, the AaNRP dsRNA was intrathoracically inoculated into female mosquitoes.The mosquitoes inoculated with GFP dsRNA served as negative controls.Three days after administration of dsRNA, the mosquitoes were intrathoracically infected with ZIKV.At 8 days after infection, these mosquitoes were allowed to bite A129 mice.Silencing the AaNRP gene did not influence the mosquito's probing time, efficiency of blood engorgement, or capacity for salivation (Appendix Given that resveratrol may modulate innate immune pathways, we next assessed whether oral administration of resveratrol in our study may influence immune hemostasis in resting skin and blood by using a flow cytometry assay.Dietary administration of resveratrol did not regulate the frequency of myeloid cells in resting skin (Appendix Fig S5A) or blood (Appendix Fig S5B).We next assessed the potential effects of resveratrol on host antiviral innate immune pathways during ZIKV infection.The skin, lymph nodes and spleens from ZIKV-infected mice at 24 hours post infection with or without dietary administration of resveratrol were collected for an RNA-Seq assay.Oral treatment with resveratrol did not modulate the expression of most of the antiviral immune genes in murine footpad skin, lymph node or spleen (Appendix Table S3).These new results have been added to revised manuscript (Line 413-416, Page 15).In this study, we identified AaNRP as a mosquito salivary protein that causes a significant influx of neutrophils and subsequent recruitment of myeloid cells toward mosquito bite sites, thus promoting arboviral infection in their target cells.

#1L. The specificity of the new anti-AaNRP antibody needs to be defined for data in
In addition, previous studies have identified other mosquito salivary components that facilitate arboviral infection.For example, an A. aegypti salivary 34-kDa protein suppresses interferon signaling to enhance DENV replication in human keratinocytes As mentioned by the reviewer in Question #1R, the previous investigation has highlighted that chemokine expression alone is not sufficient to mediate saliva-induced enhancement of arbovirus infection (Lefteri et al., 2022, Proceedings of the National Academy of Sciences of the United States of America, 119: e2114309119) and emphasized that induction of chemokines is definitely one of the essential steps in the saliva enhancement of virus infection.Based on our findings and other studies, we concluded that these salivary components may play critical roles in regulating the different steps in the lineage cascade of cutaneous infection by arboviruses.Interruption of each step in the cascade may effectively impair the whole process of cutaneous infection.#1R.Discussion-Furthermore, the discussion as written here suggests that chemokine expression is the limiting step required for saliva-enhancement of virus infection.Some other reports have suggested chemokine expression alone is not sufficient to mediate saliva-induced enhancement of arbovirus infection (e.g Lefteri 2022 that is widely cited here).Chemokine expression is clearly key (as shown here) but is one of a number of steps, and this should be placed into context in the discussion.#1R Answer: Thank you for the reviewer's suggestion.We have highlighted that chemokine expression alone is not sufficient to mediate saliva-induced enhancement of arbovirus infection (Lefteri et al., 2022, Proceedings of the National Academy of Sciences of the United States of America, 119: e2114309119) and emphasized that induction of chemokines is one of the essential steps in the saliva enhancement of virus infection (Line 480-484, Page 18).

Responses to Referee #2: #2A. Fig 1C: This experiment could include additional controls beyond PBS. For example, does heat-treatment of the protein sample prior to injection in mice negate the neutrophil influx?
#2A Answer: Thank you for the reviewer's question.Reviewer 3 raised a similar concern for the negative controls (Question #3C).According to the suggestions, we included PBS and a heat-inactivated AaNRP (hiAaNRP) as negative controls, and a salivary protein encoded by AAEL009524 acted as an unrelated control.Thus, 100 ng of each protein or an equal volume of PBS was injected into mouse footpads.The injected skin was later collected for a flow cytometry analysis.Skin injected with PBS, hiAaNRP or the AAEL009524-encoded protein showed similar levels of neutrophils (

#2B. Fig 1F-1H. The 1A8 antibody targets both Ly-6C and Ly-6G and thus depletes multiple cells type including monocytes. This experiment is not sufficient to conclude that an influx of neutrophils is a prerequisite for recruitment of other myeloid cells. The authors should perform more specific cell depletion studies to support this conclusion and evaluate additional cell populations to demonstrate the specificity of the depletion. #2B Answer:
We recognize the reviewer's concern.Nonetheless, the 1A8 monoclonal antibody (BioxCell, Cat# BP0075-1) binds mouse Ly6G specifically.Unlike the RB6-8C5 antibody, the 1A8 antibody specifically detects murine Ly6G, rather than cross-reactivity with Ly6C (https://bioxcell.com/invivoplus-anti-mouse-ly6g-bp0075-1).
Thus, the 1A8 monoclonal antibody has been widely used to specifically deplete neutrophils in mice (Coffelt et al., 2015, Nature, 522: 345-348;Finisguerra et al., 2015, Nature, 522: 349-353;Pingen et al., 2016, Immunity, 44: 1455-1469).To further address this concern, we ectopically expressed murine Ly-6C in HEK293T cells and used a Ly6C-specific antibody as a positive control to test whether the 1A8 antibody could cross-react with Ly6C.The results showed that although Ly6C-transfected cells can be well labeled by Ly6C-specific antibodies, they could not be labeled by the 1A8 antibody, thereby excluding the potential binding of the 1A8 antibody to Ly6C (Fig R1).

#2C. Fig 4G-H. These findings suggest that TLR4 is an important recognition receptor for AaNRP. Showing that AaNRP-mediated induction of chemokines is resistant to inhibition by polymyxin B would further support the authors conclusions, as the presence of LPS is a concern. #2C Answer:
We recognize the reviewer's concern.In this study, the results indicated that AaNRP activates MyD88-dependent canonical NF-κB signaling, which drives the expression of neutrophil chemoattractants and the subsequent recruitment of myeloid cells.The recombinant AaNRP protein was expressed and purified in Drosophila S2 cells.To address the reviewer's concern, we first used polymyxin B, a reagent that effectively neutralizes LPS, to assess the possibility of LPS-mediated activation of the MyD88-dependent NF-κB pathway.Purified AaNRP and LPS were incubated with sterile water (control) or with 30 μg/ml polymyxin B (neutralization) at 4 °C for 24 hours.Then, sterile water or polymyxin B-treated AaNRP and LPS were added to murine RAW264.7 macrophages for incubation.Four hours later, the cells were collected for qPCR detection of CXCL1/2/3 expression.The results showed that both sterile water-treated AaNRP and LPS significantly induced the expression of CXCL1/2/3.Polymyxin B preincubation had no influence on AaNRP-induced expression of CXCL1/2/3 but completely abolished LPS-induced CXCL1/2/3 expression (Appendix Fig S2B).These data further validated the specific induction of these chemokines by AaNRP and well addressed the concern of LPS contamination in AaNRP preparation.We have added the data to the revised manuscript (Line 282-292, Page 11).To address the reviewer's concern, we assessed the LPS contamination in the preparation of 1 μg/μl AaNRP and the PBS solution (Gibco, Cat# 10010023) used to dilute AaNRP preps by the Pierce LAL Chromogenic Endotoxin Quantitation Kit (Cat# A39552S).The endotoxin concentration in the preparation of purified AaNRP protein showed no difference with the PBS solution (average level: AaNRP 0.038 EU/ml, PBS 0.037 EU/ml, p=0.66) (Appendix Fig S2A).It is generally believed that an endotoxin content below 0.1 EU/ml has no detectable effect on the cultured cells or animals.In this case, the observed effects should be attributed to AaNRP rather than LPS contamination.5C could be more informative if the percentage and total number of infected cells within the specific cell type evaluated was reported.That is, among macrophages, did the frequency and total number of infected macrophages increase with AaNRP?Same question for other cell types.#2D Answer: We appreciate the reviewer's suggestion.We found that AaNRP did not increase the frequency of ZIKV-infected myeloid cells within their specific cell types (Fig R2A -C).Nevertheless, considering that AaNRP induced considerable recruitment of myeloid cells, and the total number of skin cells is relatively stable, the absolute number of ZIKV-infected myeloid cells was surely increased.Therefore, we believe that increases in the proportions of ZIKV-infected myeloid cells within all skin cells should be better to reflect the infection-enhancing effect of AaNRP.

#2E. For Fig 5D and other measurements of viremia, the authors should also assess the amount of infectious virus present, as the approach used could be influenced by differences in circulating cells between the experimental groups. In addition, infectious virus in the blood is more informative. Similarly, due to the changes in cell composition
in tissues (e.g.,shown in Fig 5B and 5C), using the ZIKV/GAPDH mRNA ratio to quantify viral tissue burden in murine tissues throughout this study could be misleading.#2E Answer: According to the reviewer's suggestion, we reassessed the viremia and viral loads in murine tissues throughout our study by focus forming assay.Please see Responses to Referee #3: #3A.Line 125 and Figure 1C.The authors screened the panel of proteins for neutrophil recruitment activity by inoculating these proteins subcutaneously into mouse footpads.However, the mosquito proboscis only probes the dermis in humans and cannot extend into the subcutaneous layer.Could there be differences in how antigen presenting cells resident in the dermis or even keratinocytes respond to these proteins and hence influence downstream neutrophil recruitment?The experimental approach could thus have underestimated the number of salivary proteins that could play a role in augmenting ZIKV and DENV infection.#3A Answer: We recognize the reviewer's concern.Subcutaneous injection of mosquito salivary proteins and/or mosquito-borne viruses in mouse footpads has been a well-established method to simulate mosquito salivation/bite-mediated  al., 2020al., , Lancet, 395: 1998al., -2007)).Martin-Martin et al. injected SGE via footpad injection, and they described the footpad injection as subcutaneous/intradermal injection (Martin- Martin et al., 2022, Cell reports, 39: 110648).We performed the footpad injection at a very superficial layer, which is anatomically located in the dermis layer or the transition region between the dermis and hypodermis, where the mosquito mouthparts can completely reach (Demeure et al., 2005, J Immunol, 174: 3932-3940).Thus, our footpad injection is actually intradermal or intradermal/subcutaneous, which could simulate natural mosquito bites.We have corrected all the descriptions of footpad injection as intradermal in the revised manuscript to avoid confusion.#3B.What is the normal function of AaNRP?Obviously, neutrophil recruitment is not required for successful completion of bloodmeal for mosquitoes to lay their eggs.#3B Answer: Thank you for the reviewer's question.AaNRP, an A. aegypti salivary protein encoded by AAEL007923, has not been investigated by any previous functional study.We named this protein according to its capability to cause the recruitment of neutrophils to mosquito bite sites.Intriguingly, AaNRP was specifically expressed in the salivary glands of female A. aegypti (Fig EV1F) rather than in those of male A. aegypti (Fig EV1G).Moreover, the expression of AaNRP was induced following blood feeding (Fig EV1H), suggesting a role of AaNRP in the process of blood feeding and/or host-mosquito interactions.Thus, we plan to further investigate the extended functions of AaNRP in future investigations.#3C.For many of the experiments, AaNRP was compared against PBS as a negative control.PBS is not the ideal control.A more appropriate negative control would be another protein, preferably a protein that is also excreted in Ae aegypti saliva.Given the many negative findings in Figure 1C, a negative control could have been chosen from that list of proteins.Such a control would have supported the specificity of AaNRP in mediating the effects shown in the rest of the manuscript.#3C Answer: Thank you for the reviewer's suggestion.Reviewer 2 raised a similar concern for the negative controls (Question #2A).According to the suggestions, we included PBS and a heat-inactivated AaNRP (hiAaNRP) as negative controls, and a noninflammtory control (NIC) salivary protein encoded by AAEL009524 acted as an unrelated control.Thus, 100 ng of each protein or an equal volume of PBS was injected into mouse footpads.The injected skin was later collected for a flow cytometry analysis.Skin injected with PBS, hiAaNRP or the AAEL009524-encoded protein (NIC) showed similar levels of neutrophils (Fig 1D)

#3D. Lines 138-140 and Fig S1G. Increased expression of AaNRP after a bloodmeal does not support the notion proposed in this study, that AaNRP is necessary to augment infection. AaNRP should be measured before and not after a bloodmeal.
This could be done by quantifying AaNRP expression from when the mosquito hatches to when it is ready to become gravid through a bloodmeal.#3D Answer: We agree with the reviewer's suggestion.These data (Fig EV1H) showed that AaNRP was upregulated in the mosquito salivary glands after a blood meal.Due to the infection-enhancing effect of AaNRP, blood-fed mosquitoes with upregulated AaNRP might thus be more effective in transmitting viruses to vertebrate hosts.Based on the editor's suggestion, we have now adjusted the interpretation to indicate that AaNRP might have additional functions post bloodmeal.We have added this to the revised manuscript (Line 146-148, Page 6).

#3E.
Lines 200-201.The approach of using scRNAseq to identify mediators of neutrophil recruitment completely misses a major contributor of acute inflammationpre-formed mast cell mediators.These pre-formed chemicals are stored in granules that, upon mast cell activation, would be released through degranulation, several of which are known to play roles in neutrophil recruitment.Post-mosquito bite scRNAseq would thus miss many factors that facilitate neutrophil recruitment.#3E Answer: We recognize this careful concern.The preformed chemicals stored in granules may be released from activated mast cells, thus leading to the recruitment of neutrophils (Demeure et al., 2005, J Immunol, 174: 3932-3940).scRNA-Seq was used to detect the regulation of gene transcripts post mosquito bites, thereby missing the regulation of pre-formed factors mediated by mosquito bites.We therefore assessed the activation of cutaneous mast cells at 4 hours post mosquito bite (hpb).The percentage of activated mast cells, defined as CD63+CD203c+ (Ebo et al., 2021, The Journal of allergy and clinical immunology, 147: 1143-1153), showed no significant difference between resting skin and mosquito bitten skin (Fig EV3C ), indicating that the mosquito bite does not induce the activation of cutaneous mast cells at the early time point after biting (Fig EV3C).In this study, we found that skin resident macrophages are activated by mosquito bites to release neutrophil chemoattractants.Antibody-mediated depletion of skin resident macrophages significantly offset the recruitment of neutrophils toward the mosquito bite site.These findings indicate that skin-resident macrophages play a pivotal role in neutrophil recruitment induced by mosquito bites.2E and F. The data in Figure 2E is incongruent with 2F, the latter showing that anti-F4/80 mAb treatment inhibited neutrophil infiltration compared to isotype IgG control treatment but not to baseline resting levels.As Figure 2E only showed one point per animal, it would be important to know how the data was obtained.How many sections were examined with IHC per animal?How many fields on each slide were viewed and hence sampled to calculate % neutrophil infiltration.Without this information, Figure 2E  #3G.Line 230 and 235.There is no evidence that AaNRP is "the primary" salivary protein involved in neutrophil recruitment.Neither is there any evidence that it was the "major" factor in upregulation of neutrophil chemoattractants.The authors did not assess the other salivary proteins beyond the first screen which was done through subcutaneous and not intradermal inoculation.Please rephrase.#3G Answer: Thank you for the reviewer's suggestion.We have now rephrased the expression properly to define the function of AaNRP in the revised manuscript (Line 246 and 251, Page 9).We have clarified the subcutaneous/intradermal injection details in the #3A Answer.#3H.Lines 237-240.Does silencing of AaNRP have off-target effects on mosquito salivary production/secretion? #3H Answer: We appreciate this concern and have performed a forced salivation assay to check whether AaNRP silencing affects mosquito salivation.In this experiment, the AaNRP dsRNA was intrathoracically inoculated into mosquitoes.The mosquitoes inoculated with GFP dsRNA served as negative controls.Three days post injection with dsRNA, the mosquitoes were intrathoracically injected with ZIKV.At 8 days postinjection with ZIKV, saliva from each mosquito was collected and quantified.The results showed that AaNRP silencing by dsRNA had no influence on mosquito salivary production (Appendix Fig S4C).#3I.Section under lines 320-321 and Figure 5. Inclusion of a control constituting T6167923 treatment followed by ZIKV infection without AaNRP would have been helpful.Blocking MyD88 signaling could impact ZIKV and DENV infection and thus confound the data reported in this section.#3I Answer: We recognize this concern and have included a PBS + ZIKV + T6167923 group in Fig 5B-I in the revised manuscript.Consistent with previous results, the reproduced experiments showed that AaNRP significantly enhanced myeloid cell influx (Fig 5B ) and ZIKV infection (Fig 5C-I), while the MyD88 inhibitor T6167923 efficiently attenuated these effects by AaNRP.Notably, in the absence of AaNRP, T6167923 alone had no influence on myeloid cell influx or ZIKV infection (Fig. 5B-5I).Indeed, this is understandable because mice were only i.p. injected with T6167923 once (one hour prior to s.c.injection with PBS/ZIKV/AaNRP) throughout the experiments, such a single dose of T6167923 may only specifically affect early surged inflammatory responses induced by AaNRP and may have little or no influence on ZIKV infection per se.

#3J. Lines 353-354 and Figure 6C. RT-qPCR alone to measure viral load is not
sufficient.There is no information on how AaNRP silencing would affect ZIKV lifecycle and maturation.Besides measuring viral RNA, the amount of infectious ZIKV particle in the mosquito saliva should also be shown.#3J Answer: According to the reviewer's suggestion, we reassessed the ZIKV load in mosquito saliva by a focus forming assay.Saliva from each mosquito was collected at 8 days post infection with ZIKV, and the infectious viral titers were quantified by focus forming assay.The results showed that AaNRP silencing did not influence ZIKV loads in mosquito saliva.This data has been added to Fig 6D in the revised manuscript.#3K.Lines 437-456.Resveratrol has pleiotropic effects, some of which, such as increased risk of DNA damage, are harmful.For this compound to be used prophylactically to reduce the level of viremia and hence risk of severe disease, persons living in endemic countries will have to take this drug over long periods.How would the prophylactic benefits weigh against the risk associated with long-term usage of this compound?A more balanced discussion would be helpful.#3K Answer: We recognize the reviewer's concern.Some previous studies have suggested that resveratrol can increase DNA damage in pathogenic bacteria Nonetheless, we should be aware of the potential side effects of resveratrol.Long-term epidemiological surveillance is needed to assess the safety and prophylactic benefits to prevent flavivirus transmission.We have added this discussion to the revised manuscript (Line 508-529, Page19).#3L.Lines 458-460.The notion that lower viremia will reduce human-mosquito-human transmission is incorrect (Duong et al, PNAS 2015;112:14688).Please rephrase.#3L Answer: Thank you for this correction.The relevant statements have been removed in the revised manuscript.
26th Jan 2024 1st Revision -Editorial Decision Dear Gong, Thank you for submitting a revised version of your manuscript.Your study has now been seen by two of the original referees, who find that their previous concerns have been addressed and now recommend acceptance of the manuscript.
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2nd Feb 2024 2nd Revision -Editorial Decision
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see #1N Answer and #3H Answer.4)Better quantification of infected cells and the amount of the infectious virus.Please see #1M Answer, #2D Answer, #2E Answer and #3J Answer.
1D) or monocyte-lineage myeloid cells (Fig 1E), while AaNRP-injected skin manifested much higher levels of neutrophils (Fig 1D) and monocyte-lineage myeloid cells (Fig 1E), indicating the specific role of AaNRP in inducing myeloid cell influx.Since AaNRP rather than LPS can be heat-inactivated, the result further excludes the contamination of LPS in the AaNRP preparation.#1B.Lines 71-72.There are a number of factors in mosquito saliva that have been identified as able of modulating host susceptibility to arbovirus infection, as listed later in discussion.I would suggest rephrasing this here to make this clear.#1B Answer: According to the reviewer's suggestion, we have rephrased the role of previously identified mosquito saliva factors in modulating host susceptibility to arbovirus infection (Line 70-74, Page 3).
Figure 5, which only shows 24-hour post infection.#1G Answer: We recognize the reviewer's concern.We repeated these experiments in Fig 1E, 1H, 1L and Fig 5B and assessed the influx of monocytes, macrophages and DCs at both 6 and 24 hours post injection.Overall, monocytes, macrophages, and immature dendritic cells (DCs) were highly aggregated at the AaNRP inoculation sites at both 6 hpi (Fig 1E) and 24 hpi (Fig EV2A).Inoculation of anti-neutrophil 1A8 mAb effectively blocked the recruitment of monocytes, macrophages, and immature DCs toward the AaNRP-inoculated mouse footpads at 6 hpi (Fig 1H).The bite-mediated recruitment of monocyte-lineage immune cells was impaired by AaNRP silencing at both 6 hpb (Fig 1L) and 24 hpb (Fig EV2B).The proportions of cutaneous myeloid cells increased in the AaNRP-treated footpads at 6 hpi (Fig 5B), as did the ratios of ZIKV-positive myeloid cells at 24 hpi (Fig 5C).The data have been updated in the revised manuscript.
Fig 1).Nonetheless, we validated the role of AaNRP-mediated influx of myeloid cells in flaviviral transmission by mosquitoes (Fig 5, Fig 6 and Fig 7).According to the reviewer's suggestion, the controls of resting murine skin have been added to the revised manuscript (Fig 5B).
Fig 4B/C to meaningful.As for most of the paper, the wrong statistical test has been used; ANOVA is the correct test if there are more than two groups.#1 L Answer: We validated the specificity of the new anti-AaNRP antibody by using a flow cytometry assay.The results showed that incubation of the vector-transfected 293T cells with AaNRP Ab (primary Ab) + Fluo.Ab (secondary Ab) showed a few background labeling of the cells, while incubation of the AaNRP-transfected 293T cells with AaNRP Ab + Fluo.Ab showed significantly increased labeling of the cells (Appendix FigS3A and B).These results validate the specificity of the AaNRP Ab and have been added to the revised manuscript (Line 335-337, Page 12).We have also reanalyzed the data by using one-way ANOVA (Fig4C).#1M.Figure 5-Representative gating of ZIKV positive cells should be shown.#1 M Answer: We have shown the representative gating of ZIKV-positive cells in the revised manuscript (Fig EV4B).#1N.Infected cells do not necessarily lead to release of new infectious virus and could also represented phagocytised viral debris.The ability of skin isolated infected leukocytes to release new infectious virus should be defined.#1N Answer: To address the reviewer's concern in Fig 5D, we validated the ability of skin CD11b + leukocytes isolated from the murine footpad skin coinoculated with ZIKV Fig S4A-C).Moreover, saliva from individual mosquitoes was collected by forced salivation (Miller et al., 2021, Insects, 12) at 8 days after infection.Compared to that of controls, dsRNA-mediated knockdown of AaNRP did not influence the ZIKV titer in the saliva of infected mosquitoes, which was assessed by a focus forming assay (Fig 6D).We have added the data to the revised manuscript (Line 390-395, Page 14).#1P. Figure 7-It is not clear how specific resveratrol is on innate immune responses.Ability of this agent to suppress other TLR4 activating ligands should ideally be defined as a positive control.Resveratrol could be modulating numerous innate immune pathways, and/or myeloid cell frequency in resting skin/blood, independent of AaNRP function.What effect does resveratrol have on anti-viral innate immune pathways e.g.type I IFN and IFN stimulated gene expression?All of these pathways could impact on overall host susceptibility to infection.#1P Answer: We recognize the reviewer's concern.We exploited LPS, a well-known activating ligand of the MyD88-NF-kB signaling pathway, for subcutaneous inoculation in murine footpad skin.Dietary administration of resveratrol attenuated the LPS-mediated activation of MyD88-NF-kB signaling in murine skin (Appendix Fig S6).Consistently, accumulating evidence has shown that resveratrol can efficiently inhibit the activation of TLR-MyD88-NF-kB signaling by various stimulants, including LPS, in both in vitro and in vivo conditions (Huang et al., 2021, The Journal of nutritional biochemistry, 88: 108552; Shi et al., 2017, Annals of the New York Academy of Sciences, 1403: 38-47; Zhang et al., 2014, Molecular medicine reports, 10: 101-106; Zhou et al., 2018, Molecular medicine reports, 17: 1269-1274).

#
1Q. Discussion-The results presented here suggest that AaNRP accounts for almost all virus enhancing ability of mosquito saliva.What does this mean for the role of other mosquito salivary proteins, including the finding that AaVA-1 (Sun et al) was pivotal for this process, previously published by this group?#1Q Answer: Mosquito saliva has been demonstrated to promote the infection of mosquito-borne viruses by affecting different steps of infection and by various mechanisms (Pingen et al., 2017, Trends in parasitology, 33: 645-657; Wang et al., 2023, Insect science).Accumulating evidence indicates that mosquito bites result in an influx of inflammatory neutrophils that coordinate a localized innate immune response to recruit numerous virus-permissive myeloid cells toward bite sites (Guerrero et al., 2022, Nature communications, 13: 7036; Hastings et al., 2019, Journal of virology, 93; Pingen et al., 2016, Immunity, 44: 1455-1469).Notably, neutrophil depletion and therapeutic blockade of cutaneous inflammation abrogated the bite-mediated promotion of arbovirus infection at both the skin inoculation site and lymphoid tissues (Pingen et al., 2016, Immunity, 44: 1455-1469).
or monocyte-lineage myeloid cells (Fig 1E), while AaNRP-injected skin manifested much higher levels of neutrophils (Fig 1D) and monocyte-lineage myeloid cells (Fig 1E), indicating the specific role of AaNRP in inducing myeloid cell influx.
could be due to biased sampling.#3F Answer: We recognize the reviewer's concern.Indeed, these figures represent different experiments, with Fig 2E displaying flow cytometry data and Fig 2F presenting IHC images.In light of this, we have now included a more representative IHC panel in Fig 2F in the revised manuscript.We had two footpad sections for each animal and three random scopes were sampled from each section, and the most representative image was selected to delegate the mouse (this is now clearly stated in Fig 2F legend).

I
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Table S1
In consideration of the traits of mosquito saliva, we chose the optimal dosage of inoculated salivary proteins according to previous evidence.Schmid et al. reported a total protein amount of approximately 370-600 ng in the SGE per individual mosquito (Schmid -Line 123 " We next assessed the role of these salivary proteins in triggering neutrophil influx into the host skin.Thus, 100 ng of purified salivary proteins were individually inoculated into a mouse footpad in a subcutaneous manner".It is not clear why 100ng was chosen and whether this reflects the mass inoculated by a probing mosquito.The amount salivated by mosquitoes, e.g.following forced salivation into oil or media should be define.#1D Answer: The dosage for studying mosquito saliva proteins is varied in different studies.For instance, Jin et al. utilized 2 μg of LTRIN per mouse to examine the enhancing effect of mosquito salivary proteins on ZIKV infection (Jin et al., 2018, Nature immunology, 19: 342-353).Ryuta Uraki et al. used 10 μg of AgBR1 per mouse for their investigation (Uraki et al., 2019, Nature microbiology, 4: 948-955).Martin-Martin et al. employed 6 μg of salivary gland extract (SGE) per mouse to study the impact of mosquito salivary proteins on leukocyte recruitment (Martin-Martin et al., 2022, Cell reports, 39: 110648).

(Davies et al., 2013, Nature immunology, 14: 986-995; Kashem et al., 2017, Annual review of immunology, 35: 469-499).
Nonetheless, the scRNA-Seq data indicate that Langerhans cells show a relatively low prevalence in murine skin compared to that of skin macrophages, which is consistent with previous reports (

(Davies et al., 2013, Nature immunology, 14: 986-995; Wynn et al., 2013, Nature, 496: 445-455).
According to the reviewer's suggestion, we have added this to the discussion in the revised manuscript (Line 549-559, Page 20 and 21).Figure3and 4 are generally well done, although would be more meaningful if LPS contamination of AaNRP preparation was ruled out.Confirmation of TLR4-MyD88 signalling pathway activation in primary cultures of Macrophages is required, as immortalised RAW cells often generate data that is hard to replicate in terminally differentiated (and thereby relevant) cells.

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