SUMMARY: West Nile Virus was introduced into the Western Hemisphere during the late summer of 1999 and has been causing significant and sometimes severe human diseases since that time. This article briefly touches upon the biology of the virus and provides a comprehensive review regarding recent discoveries about virus transmission, virus acquisition, and human infection and disease.

West Nile (WNV), dengue (DENV) and yellow fever (YFV) viruses are (re)emerging, mosquito-borne flaviviruses that cause human disease and mortality worldwide. Alterations in mosquito gene expression common and unique to individual flaviviral infections are poorly understood. Here, we present a microarray analysis of the Aedes aegypti transcriptome over time during infection with DENV, WNV or YFV. We identified 203 mosquito genes that were ≥5-fold differentially up-regulated (DUR) and 202 genes that were ≥10-fold differentially down-regulated (DDR) during infection with one of the three flaviviruses. Comparative analysis revealed that the expression profile of 20 DUR genes and 15 DDR genes was quite similar between the three flaviviruses on D1 of infection, indicating a potentially conserved transcriptomic signature of flaviviral infection. Bioinformatics analysis revealed changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. We also demonstrate that virally-regulated gene expression is tissue-specific. The overexpression of several virally down-regulated genes decreased WNV infection in mosquito cells and Aedes aegypti mosquitoes. Among these, a pupal cuticle protein was shown to bind WNV envelope protein, leading to inhibition of infection in vitro and the prevention of lethal WNV encephalitis in mice. This work provides an extensive list of targets for controlling flaviviral infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses. Dengue (DENV), West Nile (WNV) and Yellow Fever (YFV) viruses are responsible for severe human disease and mortality worldwide. There is no vaccine available for dengue or West Nile virus and no specific antiviral is available for any of these viral infections. These viruses are transmitted to humans through the bite of a mosquito vector. Understanding the effects of viral infection on gene expression in the mosquito is crucial to the development of effective antiviral treatments for mosquitoes and may enable researchers to interrupt the human-insect infection cycle. Here we investigate the alterations in gene expression across the entire Aedes aegypti genome during infection with DENV, YFV and WNV over time. We describe several genes that share a similar expression profile during infection with all three viruses. We also use a WNV mosquito cell, mosquito and mouse model to show that virally downregulated genes are inhibitory to infection when overexpressed and that viral regulation of mosquito genes is tissue-specific. Our results provide an extensive amount of data highlighting viral gene targets in the mosquito during infection. This data may also be used to develop broad-spectrum anti-flaviviral treatments in mosquitoes.

Dengue virus (DENV), a flavivirus of global importance, is transmitted to humans by mosquitoes. In this study, we developed in vitro and in vivo models of saliva-mediated enhancement of DENV infectivity. Serine protease activity in Aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. A serine protease inhibitor reduced saliva-mediated enhancement of DENV in vitro and in vivo , marked by a 100-fold reduction in DENV load in murine lymph nodes. A saliva-mediated infectivity enhancement screen of fractionated salivary gland extracts identified serine protease CLIPA3 as a putative cofactor, and short interfering RNA knockdown of CLIPA3 in mosquitoes demonstrated its role in influencing DENV infectivity. Molecules in mosquito saliva that facilitate viral infectivity in the vertebrate host provide novel targets that may aid in the prevention of disease.

Many arboviral diseases are uncontrolled, and the viruses that cause them are globally emerging or reemerging pathogens that produce significant disease throughout the world. The increased spread and prevalence of disease are occurring during a period of substantial scientific growth in the vector-borne disease research community. This growth has been supported by advances in genomics and proteomics, and by the ability to genetically alter disease vectors. For the first time, researchers are elucidating the molecular details of vector host-seeking behavior, the susceptibility of disease vectors to arboviruses, the immunological control of infection in disease vectors, and the determinants that facilitate transmission of arboviruses from a vector to a host. These discoveries are facilitating the development of novel strategies to combat arboviral disease, including the release of transgenic mosquitoes harboring dominant lethal genes, the introduction of arbovirus-blocking microbes into mosquito populations, and the development of acquisition- and transmission-blocking therapeutics. Understanding the role of the vector in arbovirus transmission has provided critical practical and theoretical tools to control arboviral disease.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes dengue fever in humans, worldwide. Using in vitro cell lines derived from Aedes albopictus and Aedes aegypti, the primary vectors of DENV, we report that DENV2/DENV3-infected cells secrete extracellular vesicles (EVs), including exosomes, containing infectious viral RNA and proteins. A full-length DENV2 genome, detected in arthropod EVs, was infectious to naïve mosquito and mammalian cells, including human-skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed mosquito EVs with a size range from 30 to 250 nm. Treatments with RNase A, Triton X-100, and 4G2 antibody-bead binding assays showed that infectious DENV2-RNA and proteins are contained inside EVs. Viral plaque formation and dilution assays also showed securely contained infectious viral RNA and proteins in EVs are transmitted to human cells. Up-regulated HSP70 upon DENV2 infection showed no role in viral replication and transmission through EVs. In addition, qRT-PCR and immunoblotting results revealed that DENV2 up-regulates expression of a mosquito tetraspanin-domain–containing glycoprotein, designated as Tsp29Fb, in A. aegypti mosquitoes, cells, and EVs. RNAi-mediated silencing and antibody blocking of Tsp29Fb resulted in reduced DENV2 loads in both mosquito cells and EVs. Immunoprecipitation showed Tsp29Fb to directly interact with DENV2 E-protein. Furthermore, treatment with GW4869 (exosome-release inhibitor) affected viral burden, direct interaction of Tsp29Fb with E-protein and EV-mediated transmission of viral RNA and proteins to naïve human cells. In summary, we report a very important finding on EV-mediated transmission of DENV2 from arthropod to mammalian cells through interactions with an arthropod EVs-enriched marker Tsp29Fb. Significance So far, no studies have reported whether dengue virus uses arthropod extracellular vesicles (EVs) for its transmission from vector to the mammalian host. Our study reports a very significant finding on EV-mediated transmission of dengue viruses (serotypes 2 and 3) from mosquito to mammalian, including human cells, through interactions with an arthropod EV-enriched tetraspanin domain-containing glycoprotein, Tsp29Fb.

Aedes aegypti is the primary vector of several medically relevant arboviruses including dengue virus (DENV) types 1–4. Ae. aegypti transmits DENV by inoculating virus-infected saliva into host skin during probing and feeding. Ae. aegypti saliva contains over one hundred unique proteins and these proteins have diverse functions, including facilitating blood feeding. Previously, we showed that Ae. aegypti salivary gland extracts (SGEs) enhanced dissemination of DENV to draining lymph nodes. In contrast, HPLC-fractionation revealed that some SGE components inhibited infection. Here, we show that D7 proteins are enriched in HPLC fractions that are inhibitory to DENV infection, and that recombinant D7 protein can inhibit DENV infection in vitro and in vivo. Further, binding assays indicate that D7 protein can directly interact with DENV virions and recombinant DENV envelope protein. These data reveal a novel role for D7 proteins, which inhibits arbovirus transmission to vertebrates through a direct interaction with virions. Dengue virus (DENV) is transmitted to humans by Aedes aegypti during the blood feeding process. During blood feeding, DENV and saliva proteins are inoculated into human skin. D7 proteins are prevalent and immunogenic proteins present in Ae. aegypti saliva, and assist the blood feeding process by scavenging biogenic amines. Previous data suggests that antibodies against D7 protein from Culex spp. can increase West Nile virus infection. We hypothesized that D7 proteins may also have antiviral activity. Here, we show that recombinant Ae. aegypti D7 protein can inhibit DENV infection in vitro and in vivo, and that D7 can bind to DENV virions.