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.

Vesicular stomatitis (VS) is a viral disease of veterinary importance, enzootic in tropical and subtropical regions of the Americas. In the U.S., VS produces devastating economic losses, particularly in the southwestern states where the outbreaks display an occurrence pattern of 10-year intervals. To date, the mechanisms of the geographic spread and maintenance cycles during epizootics remain unclear. This is due, in part, to the fact that VS epidemiology has a complex of variables to consider, including a broad range of vertebrate hosts, multiple routes of transmission, and an extensive diversity of suspected vector species acting as both mechanical and biological vectors. Infection and viral progression within vector species are highly influenced by virus serotype, as well as environmental factors, including temperature and seasonality; however, the mechanisms of viral transmission, including non-conventional pathways, are yet to be fully studied. Here, we review VS epidemiology and transmission mechanisms, with comparisons of transmission evidence for the four most incriminated hematophagous dipteran taxa: Aedes mosquitoes, Lutzomyia sand flies, Simulium black flies, and Culicoides biting midges.

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 responsible for serious human disease worldwide and the World Health Organization estimates that over 2 billion people are at risk for disease. There are no vaccines or specific antiviral medications currently available for DENV infection. DENV is transmitted to humans by infected mosquitoes during feeding and probing. By examining the effects of virus infection on gene expression, and interactions between virus and vector, we may be able to find new targets for prevention and treatment. Here we look at a mosquito protein, CRVP379, whose gene expression was highly increased during DENV infection in mosquitoes. We show a requirement for CRVP379 during DENV infection in the mosquito and a correlation between the levels of CRVP379 and levels of infection. Our results indicate that the protein may be acting with a putative DENV receptor in the mosquito, prohibitin protein. These data also suggest that blocking CRVP379 function may be used to block DENV infection in the mosquito.

Norte de Santander is a region in Colombia with a high incidence of dengue virus (DENV). In this study, we examined the serum concentration of anti-Aedes salivary gland extract (SGE) antibodies as a biomarker of DENV infection and transmission, and assessed the duration of anti-SGE antibody concentration after exposure to the vector ceased. We also determined whether SGE antibody concentration could differentiate between positive and negative DENV infected individuals and whether there are differences in exposure for each DENV serotype. We observed a significant decrease in the concentration of IgG antibodies at least 40 days after returning to an “Ae. aegypti-free” area. In addition, we found significantly higher anti-SGE IgG concentrations in DENV positive patients with some difference in exposure to mosquito bites among DENV serotypes. We conclude that the concentration of IgG antibodies against SGE is an accurate indicator of risk of dengue virus transmission and disease presence.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant global human disease and mortality. One approach to develop treatments for DENV infection and the prevention of severe disease is through investigation of natural medicines. Inflammation plays both beneficial and harmful roles during DENV infection. Studies have proposed that the oxidative stress response may be one mechanism responsible for triggering inflammation during DENV infection. Thus, blocking the oxidative stress response could reduce inflammation and the development of severe disease. Garlic has been shown to both reduce inflammation and affect the oxidative stress response. Here, we show that the garlic active compounds diallyl disulfide (DADS), diallyl sulfide (DAS) and alliin reduced inflammation during DENV infection and show that this reduction is due to the effects on the oxidative stress response. These results suggest that garlic could be used as an alternative treatment for DENV infection and for the prevention of severe disease development.