Flaviviral transmission enhancer in mosquito saliva – VirSalivaEnhancer

Flaviviruses are viruses with an RNA genome that are transmitted by mosquitoes. When mosquitoes bite, they inject saliva to numb the skin. When the mosquito is infected, this saliva contains viruses that infect the skin before spreading to the rest of the body and causing symptoms. Importantly, saliva also contains compounds that promote skin infection and therefore transmission. In our study, we collected mosquito saliva to detect the RNA virus using molecular biology methods. Using these RNA quantification methods, we also discovered the presence of a viral RNA fragment and other viral RNA in saliva. We then used human skin cells and human skin explants to evaluate the effect of these salivary compounds on skin infection and therefore transmission.

We discovered two types of salivary compounds that increase skin infection during mosquito bites. On the one hand, we discovered that all flaviviruses secrete non-coding viral RNA into saliva. This non-coding viral RNA inhibits the skin's immune response, providing an advantage to viruses. On the other hand, mosquito saliva contains lipids which also promote skin infection and therefore transmission. These salivary lipids modulate the lipid composition of the skin to improve flavivirus replication. This study thus revealed the existence of two new salivary compounds which promote the transmission of all flaviviruses.

This study resulted in the publication of 10 articles. In these articles, we demonstrated the existence of non-coding viral RNA in mosquito saliva and showed its function in amplifying transmission. In several other papers, we discovered the function of mosquito lipids in transmission. The multitude of scientific articles describes how these two transmission amplification factors have multiple roles in transmission.

Furthermore, we have submitted a patent to apply these discoveries in a new control strategy.

Fundings from ANR allowed the completion of several projects and the ANR funding is mentioned in the following scientific articles:

1. Chowdhury, Avisha, Cassandra M. Modahl, Dorothée Missé, R. Manjunatha Kini, et Julien Pompon. « High Resolution Proteomics of Aedes Aegypti Salivary Glands Infected with Either Dengue, Zika or Chikungunya Viruses Identify New Virus Specific and Broad Antiviral Factors ». Scientific Reports 11, no 1 (8 décembre 2021): 1 12. doi.org/10.1038/s41598-021-03211-0

2. Vial, Thomas, Guillaume Marti, Dorothée Missé, et Julien Pompon. « Lipid Interactions Between Flaviviruses and Mosquito Vectors ». Frontiers in Physiology 12 (2021): 1744. doi.org/10.3389/fphys.2021.763195.

3. Xiang, Benjamin Wong Wei, Wilfried A. A. Saron, James C. Stewart, Arthur Hain, Varsha Walvekar, Dorothée Missé, Fréderic Thomas, Kini M., Roch B., Claridge-Chang A. St John A. et Pompon J. « Dengue Virus Infection Modifies Mosquito Blood-Feeding Behavior to Increase Transmission to the Host ». Proceedings of the National Academy of Sciences 119, no 3 (18 janvier 2022). doi.org/10.1073/pnas.2117589119

Pathogenic flaviviruses of global importance, such as dengue (DENV), Zika (ZIKV) and yellow fever (YFV) viruses, are transmitted by a mosquito bite. Mosquito saliva released during biting contain viruses that initiate skin infection, which then spreads to the rest of the body, triggering symptoms associated with morbidity and mortality. Understanding factors that determine bite-initiated skin infection will lead to protective strategies against transmission. Several teams, including ours, studied mosquito saliva components and identified a few mosquito salivary proteins with proviral function. Here, we undertake a novel approach by studying flaviviral components in saliva. In DENV2-infected saliva, we detected subgenomic flaviviral RNA (sfRNA), a non-coding flaviviral RNA with immune inhibitory functions. We further showed that sfRNA is contained in salivary exosomes that transfer viral material to skin cells. In support of a role for salivary sfRNA in transmission, we found that early presence of sfRNA increases infection by inhibiting innate immune response in a skin cell line. Our preliminary results suggest that salivary sfRNA-containing exosomes represent a novel actor in DENV2 transmission. Because all flaviviruses produce sfRNA, this mechanism of transmission enhancement may be shared across mosquito-borne flaviviruses, representing an ideal universal target to block their transmission. In the proposal, we test the hypothesis that flaviviruses exploit mosquito salivary exosomes to transfer an immune inhibitor into skin cells to reduce immune response, thereby enhancing transmission. In task 1, using a validated in vitro model, we will isolate the exosome subpopulation that contains DENV2 sfRNA and describe its proteomic, transcriptomic and lipid contents, and microscopically characterize it. We will determine which types of skin cells are targeted by these exosomes. By characterizing sfRNA-containing exosomes, task 1 will shed light on their biogenesis and functions. In task 2, we will determine the function of DENV2 sfRNA-containing exosomes in transmission in in vitro and in vivo models. We will determine the impact of early presence of sfRNA and supplementation with sfRNA-containing exosomes on innate immune response using RNA sequencing and on infection in DENV2-infected skin cells targeted by exosomes. Functional characterization of regulated genes will reveal the functions of sfRNA and exosomes, separately and together. We will then study how salivary exosome depletion in transgenic mosquitoes influences bite-initiated DENV2 infection and immune response in skin using a mouse model. We will quantify infection and immune response in skin cells early after biting by single-cell RNA sequencing. By deploying cutting-edge technologies for the first time to study mosquito biting, task 2 will decipher the function of salivary sfRNA-containing exosomes. In task 3, we will determine whether sfRNA-enhancement of skin infection is conserved in the other serotypes of DENV, in ZIKV and YFV. We will quantify sfRNA in saliva infected by these pathogenic flaviviruses, determine whether salivary sfRNA is present in exosomes and quantify the impact of each virus sfRNA on infection and immune response in exosome-targeted skin cells. Task 3 will test the generality of the mechanism to evaluate the public health potential of exosome-targeting strategies. Potential applications of our results include development of immunization strategies against salivary exosomes and the use of mosquito exosomes as vehicle to deliver therapeutics to skin. The consortium gathers expertise in molecular entomology, bioinformatics, single-cell RNA sequencing, mouse immunology, dermatology and exosome biology from a worldwide network of collaborators. The novelty of the hypothesis and the approach together with the use of cutting-edge technologies will provide the PI with the adequate platform to establish its research career.