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

Garlic Organosulfur Compounds Reduce Inflammation and Oxidative Stress during Dengue Virus Infection

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

Natural Mosquito-Pathogen Hybrid IgG4 Antibodies in Vector Borne Diseases: A Hypothesis

Chronic exposure to antigens may favor the production of IgG4 antibodies over other antibody types. Recent studies have shown that up to a 30% of normal human IgG4 is bi-specific and is able to recognize two antigens of different nature. A requirement for this specificity is the presence of both eliciting antigens in the same time and at the same place where the immune response is induced. During transmission of most vector-borne diseases, the pathogen is delivered to the vertebrate host along with the arthropod saliva during blood feeding and previous studies have shown the existence of IgG4 antibodies against mosquito salivary allergens. However, there is very little ongoing research or information available regarding IgG4 bi-specificity with regards to infectious disease, particularly during immune responses to vector-borne diseases such as malaria, filariasis or dengue virus infection. Here, we provide background information and present our hypothesis that IgG4 may not only be a useful tool to measure exposure to infected mosquito bites, but that these bi-specific antibodies may also play an important role in modulation of the immune response against malaria and other vector-borne diseases in endemic settings

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

LC+MS/MS hits from pooled inhibitory HPLC SGE fractions.

<p>LC+MS/MS hits from pooled inhibitory HPLC SGE fractions.</p

LC+MS/MS hits from SGE/DENV2 binding assay.

<p>LC+MS/MS hits from SGE/DENV2 binding assay.</p

Recombinant D7 protein binds DENV2 virions and DENV2 envelope protein.

<p>(A) An enzyme-linked immunosorbent assay was developed by coating plates with control MMP protein or recombinant D7 protein and then measuring binding of DENV2 virions. (B) An enzyme-linked immunosorbent assay was developed by coating plates with recombinant D7 protein and then measuring binding of bovine serum albumin (BSA), DENV2 virions, and recombinant DENV2 envelope protein. Interactions were detected using antibodies against DENV2 envelope protein and a secondary antibody conjugated to horse radish peroxidase. Absorbance was detected at OD 450 nm. Student’s t tests were performed to assess statistical significance between groups.</p

LC+MS/MS hits from <i>Aedes aegypti</i> saliva.

<p>LC+MS/MS hits from <i>Aedes aegypti</i> saliva.</p