The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity

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 the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission. Support for this research came from a fellowship from the McLauglin Family Foundation to TS, scholarships from the Fondation pour la Recherche Médicale (FRM project SPF202110013925) to HM, from the Institut Méditerranéen Hospitalier (IHU, Marseille) to IMSP and from the graduate school French Ministry of Higher Education and Research to FRC and FR, UTMB start-up funds and Cancer Prevention & Research Institute of Texas grant RP200650 to LJK and MKS, the Ministerio de Ciencia e Innovación (PID2020-115696RB-I00) to MAM-R, Cancer Prevention & Research Institute of Texas grant ID RR210018 to GN, Ministry of Education (Singapore) Tier3 grant (MOE2015-T3-1-003) to RMK and JP, a National Medical Research Council (Singapore) ZRRF grant (ZRRF/007/2017) to JP, a French Agence Nationale de la Recherche grant (ANR-20-CE15-0006) to JP, and the Duke-NUS Signature Research Programme funded by the Agency for Science Technology and Research (A*Star Singapore) and NIH/NIAID P01 AI150585 to MAGB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.