A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.

Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. Once the vector has become systemically and persistently infected, the vector is able to transmit the virus to an uninfected vertebrate host. In order to systemically infect the vector, the virus must cope with innate immune responses and overcome several tissue barriers associated with the midgut and the salivary glands. In this review we describe, in detail, the typical arbovirus infection route in competent mosquito vectors. Based on what is known from the literature, we explain the nature of the tissue barriers that arboviruses are confronted with in a mosquito vector and how arboviruses might surmount these barriers. We also point out controversial findings to highlight particular areas that are not well understood and require further research efforts.

Mosquitoes (Aedes aegypti) were genetically modified to exhibit impaired vector competence for dengue type 2 viruses (DENV-2). We exploited the natural antiviral RNA interference (RNAi) pathway in the mosquito midgut by constructing an effector gene that expresses an inverted-repeat (IR) RNA derived from the premembrane protein coding region of the DENV-2 RNA genome. The A. aegypti carboxypeptidase A promoter was used to express the IR RNA in midgut epithelial cells after ingestion of a bloodmeal. The promoter and effector gene were inserted into the genome of a white-eye Puerto Rico Rexville D (Higgs' white eye) strain by using the nonautonomous mariner MosI transformation system. A transgenic family, Carb77, expressed IR RNA in the midgut after a bloodmeal. Carb77 mosquitoes ingesting an artificial bloodmeal containing DENV-2 exhibited marked reduction of viral envelope antigen in midguts and salivary glands after infection. DENV-2 titration of individual mosquitoes showed that most Carb77 mosquitoes poorly supported virus replication. Transmission in vitro of virus from the Carb77 line was significantly diminished when compared to control mosquitoes. The presence of DENV-2-derived siRNAs in RNA extracts from midguts of Carb77 and the loss of the resistance phenotype when the RNAi pathway was interrupted proved that DENV-2 resistance was caused by a RNAi response. Engineering of transgenic A. aegypti that show a high level of resistance against DENV-2 provides a powerful tool for developing population replacement strategies to control transmission of dengue viruses.RNA silencing ͉ transgenesis ͉ genetic control ͉ mosquito ͉ dengue disease D engue viruses (DENV) [Flaviviridae; Flavivirus; DENV types 1-4 (DENV-1-4)] threaten public health in Ͼ100 countries and infect an estimated 50 million people annually (1, 2). The mosquito, Aedes aegypti, is the principal vector for epidemic dengue disease (3). The urban DENV transmission cycle involves only humans and mosquitoes. No DENV vaccines are currently available, and vector control strategies that minimize human-mosquito contact have largely failed (4, 5). New control strategies are needed. One possible strategy is to replace vector populations competent to transmit DENVs with pathogenincompetent vectors (6). The essential features of this genetic control strategy are to identify genes that express antiviral molecules in the vector, link this gene (or genes) to a genetic drive system [transposable elements (TE), meiotic drive, or homing endonuclease genes] and introgress the gene(s) into field populations (7-9). A key step in developing this control strategy is to identify effector genes that, when expressed in the vector, inhibit DENV replication. Proof of principle for RNA interference (RNAi)-like disruption of DENV-2 vector competence was previously demonstrated by using a nonheritable alphavirus expression system (10). Applying the principle of heritable gene silencing in transgenic Drosophila melanogaster, Caenorhabditis elegans, and A. aegypti (11-13), we gene...

Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.

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