The anti-immune dengue subgenomic flaviviral RNA is found in vesicles in mosquito saliva and associated with increased infectivity
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Curated by eLife
Evaluation Summary:
Mosquito saliva can enhance transmission of arboviruses. Here, authors demonstrated that the anti-immune non-coding RNA from Dengue virus, known as the subgenomic flavivirus RNA (sfRNA), is secreted into mosquito saliva within the extracellular vesicles and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. The study potentially expands our understanding of flavivirus transmission.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We show that salivary sfRNA is protected in detergent-sensitive, protease-resistant compartments. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in human cells. Since sfRNA potently inhibits innate immunity in human cells, we posit that sfRNA in mosquito saliva is present in extracellular vesicles that deliver it to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.
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Author Response
Evaluation Summary:
Mosquito saliva can enhance transmission of arboviruses. Here, authors demonstrated that the anti-immune non-coding RNA from Dengue virus, known as the subgenomic flavivirus RNA (sfRNA), is secreted into mosquito saliva within the extracellular vesicles and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. The study potentially expands our understanding of flavivirus transmission.
We thank the editors for the public evaluation summary and point out that while this manuscript does not answer all the questions regarding the mechanism of sfRNA delivery into cells at the bite site (which indeed is still an open question in terms of mechanisms of EV entry), it significantly enhances our understanding of what an infected mosquito …
Author Response
Evaluation Summary:
Mosquito saliva can enhance transmission of arboviruses. Here, authors demonstrated that the anti-immune non-coding RNA from Dengue virus, known as the subgenomic flavivirus RNA (sfRNA), is secreted into mosquito saliva within the extracellular vesicles and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. The study potentially expands our understanding of flavivirus transmission.
We thank the editors for the public evaluation summary and point out that while this manuscript does not answer all the questions regarding the mechanism of sfRNA delivery into cells at the bite site (which indeed is still an open question in terms of mechanisms of EV entry), it significantly enhances our understanding of what an infected mosquito deposits in the skin. Formal proof that DENV salivary sfRNA enhances transmission would require tools that are currently unavailable: (1) an animal model system that accurately reproduces transmission of DENV (at this time such a tractable experimental model could be carried out for West Nile virus in wt mice but not for DENV); (2) DENV mutants that produce very low sfRNA and are infectious in mosquitoes to permit transmission experiments.
Our study provides significant breakthroughs in our knowledge of the earliest events in DENV infection: (1) The study shows compelling data for the presence of sfRNA in a detergent-sensitive, protease-resistant compartment, (2) the manuscript presents the first visualization of viral RNAs in salivary EVs and given the quantitative nature of the imaging permit us to conclude that there is sfRNA in these EVs, and (3) the data shown lead to a strong association between levels of sfRNA and saliva infectivity. These novel findings provide important insights into salivary enhancement of transmission and given what we already know about sfRNA action, our study justifies the model proposed.
Reviewer #1 (Public Review):
The study is focused on the role of noncoding RNA (sfRNA) of DENV in mosquito transmission of the virus. The requirement of sfRNA for efficient transmission of flaviviruses by mosquitoes is well-documented, however the exact mechanisms of this effect are not clearly established. In this manuscript, authors demonstrated that DENV sfRNA is secreted into mosquito saliva within the extracellular vesicles (EV) and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. This is a novel and intriguing finding that has a potential to expand our understanding of flavivirus transmission and functions of sfRNA.
We thank the reviewer for pointing out the novelty and potential of our work to expand the understanding of flavivirus transmission.
The data provided are mostly compelling and provide answers to posed questions. However, additional evidence for EV-mediated delivery of sfRNA into acceptor human cells and the effect of this sfRNA on viral replication in acceptor cells are required to further our understanding of mechanistic aspects of how sfRNA is delivered by salivary extracellular vesicles and how it facilitates virus replication in acceptor cells. A number of additional experiments and clarifications has been requested to clarify this.
We thank the reviewer for the positive comments and useful suggestions presented in the public review.
Reviewer #2 (Public Review):
This short report by Yeh et al. reveals the presence of sfRNA in mosquito saliva and that it might enhance DENV infection in human Huh7 cells. By referring to literature, the authors propose that salivary sfRNA is secreted by EVs, and is immunosuppressive. The salivary sfRNA might facilitate DENV transmission and disease prevalence in nature.
Strength: The methods are rigorous, results are clearly presented and the manuscript is well written.
Weakness: sfRNA has long been recognized to interfere with the immune system in the flavivirus field. This study represents a modest advance. Additionally, even as a short report, the study fails to provide sufficient self-standing evidence to support its key claims. The study depends heavily on published literature to support its key conclusions.
We thank the reviewer for the positive comments about rigor and presentation of the paper, but respectfully disagree with the suggestion that this work represents a modest advance. We recapitulate that our manuscript presents the first compelling data for the presence of sfRNA in mosquito salivary EVs and provides a strong association between levels of sfRNA and saliva infectivity. The study, like much important science, stands on the shoulders of previous studies and this should not be grounds for criticism.
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Author Response:
The authors thank the editors and reviewers for their thoughtful review. We focus our response on three points the editors considered essential revisions:
The authors will work on the following:
- Providing direct evidence that DENV sfRNA from mosquito saliva is delivered into saliva-exposed human cells.
- Although the effect of sfRNA has been well established in many cell types, we will test effects on human cells found in the skin.
We expressed disagreement over a third request to test the outcomes of high sfRNA:gRNA to that of low sfRNA:gRNA infection in mice. The mouse model is not an appropriate model to test an anti-innate immune mediator in dengue and indeed results obtained with this model would not be biologically relevant. We are pleased that the editors have agreed with this rebuttal.
We believe that the data …
Author Response:
The authors thank the editors and reviewers for their thoughtful review. We focus our response on three points the editors considered essential revisions:
The authors will work on the following:
- Providing direct evidence that DENV sfRNA from mosquito saliva is delivered into saliva-exposed human cells.
- Although the effect of sfRNA has been well established in many cell types, we will test effects on human cells found in the skin.
We expressed disagreement over a third request to test the outcomes of high sfRNA:gRNA to that of low sfRNA:gRNA infection in mice. The mouse model is not an appropriate model to test an anti-innate immune mediator in dengue and indeed results obtained with this model would not be biologically relevant. We are pleased that the editors have agreed with this rebuttal.
We believe that the data presented already are very exciting and deserve publication in eLife and by further enhancing these with the first two revisions we believe the paper will be an even stronger contribution.
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Reviewer #2 (Public Review):
This short report by Yeh et al. reveals the presence of sfRNA in mosquito saliva and that it might enhance DENV infection in human Huh7 cells. By referring to literature, the authors propose that salivary sfRNA is secreted by EVs, and is immunosuppressive. The salivary sfRNA might facilitate DENV transmission and disease prevalence in nature.
Strength: The methods are rigorous, results are clearly presented and the manuscript is well written.
Weakness: sfRNA has long been recognized to interfere with the immune system in the flavivirus field. This study represents a modest advance. Additionally, even as a short report, the study fails to provide sufficient self-standing evidence to support its key claims. The study depends heavily on published literature to support its key conclusions.
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Reviewer #1 (Public Review):
The study is focused on the role of noncoding RNA (sfRNA) of DENV in mosquito transmission of the virus. The requirement of sfRNA for efficient transmission of flaviviruses by mosquitoes is well-documented, however the exact mechanisms of this effect are not clearly established. In this manuscript, authors demonstrated that DENV sfRNA is secreted into mosquito saliva within the extracellular vesicles (EV) and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. This is a novel and intriguing finding that has a potential to expand our understanding of flavivirus transmission and functions of sfRNA.
The data provided are mostly compelling and provide answers to posed questions. However, additional evidence for EV-mediated delivery of sfRNA into …
Reviewer #1 (Public Review):
The study is focused on the role of noncoding RNA (sfRNA) of DENV in mosquito transmission of the virus. The requirement of sfRNA for efficient transmission of flaviviruses by mosquitoes is well-documented, however the exact mechanisms of this effect are not clearly established. In this manuscript, authors demonstrated that DENV sfRNA is secreted into mosquito saliva within the extracellular vesicles (EV) and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. This is a novel and intriguing finding that has a potential to expand our understanding of flavivirus transmission and functions of sfRNA.
The data provided are mostly compelling and provide answers to posed questions. However, additional evidence for EV-mediated delivery of sfRNA into acceptor human cells and the effect of this sfRNA on viral replication in acceptor cells are required to further our understanding of mechanistic aspects of how sfRNA is delivered by salivary extracellular vesicles and how it facilitates virus replication in acceptor cells. A number of additional experiments and clarifications has been requested to clarify this.
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Evaluation Summary:
Mosquito saliva can enhance transmission of arboviruses. Here, authors demonstrated that the anti-immune non-coding RNA from Dengue virus, known as the subgenomic flavivirus RNA (sfRNA), is secreted into mosquito saliva within the extracellular vesicles and can facilitate infection of the acceptor human cells when delivered together with infectious virus in mosquito saliva. The study potentially expands our understanding of flavivirus transmission.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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