Arthropod EVs Mediate Dengue Virus Transmission Through Interaction With a Tetraspanin Domain Containing Glycoprotein Tsp29Fb

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

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Citation: Londono-Renteria, B., Troupin, A., Conway, M. J., Vesely, D., Ledizet, M., Roundy, C. M., . . . Colpitts, T. M. (2015). Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein. Plos Pathogens, 11(10), 23. doi:10.1371/journal.ppat.1005202Dengue 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

A prospective cohort study to assess seroprevalence, incidence, knowledge, attitudes and practices, willingness to pay for vaccine and related risk factors in dengue in a high incidence setting

Abstract Background Dengue is one of the most important vector-borne diseases in the world, causing significant morbidity and economic impact. In Colombia, dengue is a major public health problem. Departments of La Guajira, Cesar and Magdalena are dengue endemic areas. The objective of this research is to determine the seroprevalence and the incidence of dengue virus infection in the participating municipalities from these Departments, and also establish the association between individual and housing factors and vector indices with seroprevalence and incidence. We will also assess knowledge, attitudes and practices, and willingness-to-pay for dengue vaccine. Methods A cohort study will be assembled with a clustered multistage sampling in 11 endemic municipalities. Approximately 1000 homes will be visited to enroll people older than one year who living in these areas, who will be followed for 1 year. Dengue virus infections will be evaluated using IgG indirect ELISA and IgM and IgG capture ELISA. Additionally, vector indices will be measured, and adult mosquitoes will be captured with aspirators. Ovitraps will be used for continuous estimation of vector density. Discussion This research will generate necessary knowledge to design and implement strategies with a multidimensional approach that reduce dengue morbidity and mortality in La Guajira and other departments from Colombian Caribbean

Bioabsorption and Effectiveness of Long-Lasting Permethrin-Treated Uniforms Over Three Months Among North Carolina Outdoor Workers

Abstract Background Vector-borne diseases are an important cause of morbidity and mortality in the USA. Effective, convenient prevention methods are needed. Long-lasting permethrin-impregnated (LLPI) clothing can prevent tick bites, however, additional information is needed on the real-world effectiveness and safety of this preventative measure. Methods In this pilot study, we recruited state and county park employees from North Carolina to wear LLPI uniforms for three months during the summer of 2016. We collected spot urine samples for biomonitoring of permethrin metabolites at one week, one month and three months after first use of the LLPI uniform. Following three months of wear, we collected pants and socks and analyzed them for permethrin content and mortality to ticks and mosquitoes. Results Thirteen park employees were included in the analysis. Bioactive amounts of permethrin remained in all clothing swatches tested, although there was great variability. Tick mortality was high, with 78% of pant and 88% of sock swatches having mean knockdown percentages ≥ 85%. In contrast, mosquito mortality was low. Over the study period, the absorbed dosage of permethrin averaged < 4 μg/kg/d of body weight based on measurements of three metabolites. Conclusions LLPI clothing retained permethrin and bioactivity against ticks after three months of use in real-world conditions. The estimated absorbed dosage of permethrin was well below the U.S. EPA level of concern, suggesting that LLPI clothing can be used safely by outdoor workers for tick bite prevention

Malaria vector research and control in Haiti: a systematic review

BACKGROUND: Haiti has a set a target of eliminating malaria by 2020. However, information on malaria vector research in Haiti is not well known. This paper presents results from a systematic review of the literature on malaria vector research, bionomics and control in Haiti. METHODS: A systematic search of literature published in French, Spanish and English languages was conducted in 2015 using Pubmed (MEDLINE), Google Scholar, EMBASE, JSTOR WHOLIS and Web of Science databases as well other grey literature sources such as USAID, and PAHO. The following search terms were used: malaria, Haiti, Anopheles, and vector control. RESULTS: A total of 132 references were identified with 40 high quality references deemed relevant and included in this review. Six references dealt with mosquito distribution, seven with larval mosquito ecology, 16 with adult mosquito ecology, three with entomological indicators of malaria transmission, eight with insecticide resistance, one with sero-epidemiology and 16 with vector control. In the last 15 years (2000–2015), there have only been four published papers and three-scientific meeting abstracts on entomology for malaria in Haiti. Overall, the general literature on malaria vector research in Haiti is limited and dated. DISCUSSION: Entomological information generated from past studies in Haiti will contribute to the development of strategies to achieve malaria elimination on Hispaniola. However it is of paramount importance that malaria vector research in Haiti is updated to inform decision-making for vector control strategies in support of malaria elimination

An. gambiae gSG6-P1 evaluation as a proxy for human-vector contact in the Americas : a pilot study

Background: During blood meal, the female mosquito injects saliva able to elicit an immune response in the vertebrate. This immune response has been proven to reflect the intensity of exposure to mosquito bites and risk of infection for vector transmitted pathogens such as malaria. The peptide gSG6-P1 of An. gambiae saliva has been demonstrated to be antigenic and highly specific to Anopheles as a genus. However, the applicability of gSG6-P1 to measure exposure to different Anopheles species endemic in the Americas has yet to be evaluated. The purpose of this pilot study was to test whether human participants living in American countries present antibodies able to recognize the gSG6-P1, and whether these antibodies are useful as a proxy for mosquito bite exposure and malaria risk. Methods: We tested human serum samples from Colombia, Chile, and the United States for the presence of IgG antibodies against gSG6-P1 by ELISA. Antibody concentrations were expressed as delta optical density (Delta OD) of each sera tested in duplicates. The difference in the antibody concentrations between groups was tested using the nonparametric Mann Whitney test (independent groups) and the nonparametric Wilcoxon matched-pairs signed rank test (dependent groups). All differences were considered significant with a P < 0.05. Results: We found that the concentration of gSG6-P1 antibodies was significantly correlated with malaria infection status and mosquito bite exposure history. People with clinical malaria presented significantly higher concentrations of IgG anti-gSG6-P1 antibodies than healthy controls. Additionally, a significant raise in antibody concentrations was observed in subjects returning from malaria endemic areas. Conclusion: Our data shows that gSG6-P1 is a suitable candidate for the evaluation of exposure to Anopheles mosquito bites, risk of malaria transmission, and effectiveness of protection measures against mosquito bites in the Americas

Recombinant D7 protein inhibits DENV2 infection in AGB6 mice.

<p>Mice deficient in receptors for type I and type II interferons, (IFNAGR-/-, AGB6) were inoculated subcutaneously into both rear footpads with 20 μl containing either 10⁷ genome equivalents of either DENV2 alone or DENV2 plus recombinant D7 protein. Forty-eight hours post-infection, mice were euthanized and left and right foot pads and left and right popliteal draining lymph nodes (DLN) were collected independently. (A) Total RNA was harvested from footpad tissue and qRT-PCR was performed to assess relative levels of DENV2 vRNA. Relative DENV2 vRNA was normalized to actin gene expression. (B) Total RNA was harvested from DLNs and qRT-PCR was performed to assess relative levels of DENV2 vRNA. Relative DENV2 vRNA was normalized to actin gene expression. 5 mice were present in each group and the data represents a single experiment. Unpaired t tests were performed to assess statistical significance.</p