Date Published: October 22, 2015
Publisher: Public Library of Science
Author(s): Berlin Londono-Renteria, Andrea Troupin, Michael J Conway, Diana Vesely, Michael Ledizet, Christopher M. Roundy, Erin Cloherty, Samuel Jameson, Dana Vanlandingham, Stephen Higgs, Erol Fikrig, Tonya M. Colpitts, Abraham L. Brass.
Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.
Dengue virus (DENV) is the most important arbovirus in tropical areas leading to substantial pediatric morbidity and mortality worldwide [1–6]. DENV is transmitted to humans via the bite of an infected mosquito of the Aedes spp. Infection with DENV in humans can result in dengue fever (DF), dengue shock symptom (DSS) and dengue hemorrhagic fever (DHF), the latter two that can lead to severe disease and death. There are no specific antivirals or approved vaccines for use in DENV treatment or prevention [4,7–9]. Current dengue control methods rely mostly on activities to reduce vector population [10,11]. The increase in number of cases despite vector control indicates that these strategies are not as effective as expected, and that new tools need be developed to alleviate disease burden in endemic areas [12–14].
Flaviviruses are known to modify gene expression in their mosquito transmission vectors during infection. Our previous results showed that infection of Ae. aegypti with either DENV, WNV or YFV, modifies expression levels of at least 405 genes . The study of mosquito genes modified during flavivirus infection may lead to the identification of key vector antiviral mechanisms as well as key factors for interruption of the viral life cycle. One of the genes that we identified as being significantly upregulated during DENV infection was the CRVP379 gene. Cysteine-rich venom protein (CRVPs) are members of a large family of cysteine-rich secretory proteins (CRISPs), predominantly found in mammalian males and reptile venom . CRISPs contain characteristic cysteine rich C-terminal domains thought to act as ion channel regulators  and are also characterized by their role in proteolytic and defense mechanisms . CRISPs and CRVPs have been described in a broad spectrum of insects and higher vertebrates [59–62].