Andrea Troupin

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

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.

Aedes aegypti D7 Saliva Protein Inhibits Dengue Virus Infection.

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.

A relevant in vitro human model for the study of Zika virus antibody-dependent enhancement.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently been responsible for a serious outbreak of disease in South and Central America. Infection with ZIKV has been associated with severe neurological symptoms and the development of microcephaly in unborn fetuses. Many of the regions involved in the current outbreak are known to be endemic for another flavivirus, dengue virus (DENV), which indicates that a large percentage of the population may have pre-existing DENV immunity. Thus, it is vital to investigate what impact pre-existing DENV immunity has on ZIKV infection. Here, we use primary human myeloid cells as a model for ZIKV enhancement in the presence of DENV antibodies. We show that sera containing DENV antibodies from individuals living in a DENV-endemic area are able to enhance ZIKV infection in a human macrophage-derived cell line and primary human macrophages. We also demonstrate altered pro-inflammatory cytokine production in macrophages with enhanced ZIKV infection. Our study indicates an important role for pre-existing DENV immunity on ZIKV infection in primary human immune cells and establishes a relevant in vitro model to study ZIKV antibody-dependent enhancement.

Arbovirosis and potential transmission blocking vaccines

Infectious diseases caused by arboviruses (viruses transmitted by arthropods) are undergoing unprecedented epidemic activity and geographic expansion. With the recent introduction of West Nile virus (1999), chikungunya virus (2013) and Zika virus (2015) to the Americas, stopping or even preventing the expansion of viruses into susceptible populations is an increasing concern. With a few exceptions, available vaccines protecting against arboviral infections are nonexistent and current disease prevention relies on vector control interventions. However, due to the emergence of and rapidly spreading insecticide resistance, different disease control methods are needed. A feasible method of reducing emerging tropical diseases is the implementation of vaccines that prevent or decrease viral infection in the vector. These vaccines are designated ‘transmission blocking vaccines’, or TBVs. Here, we summarize previous TBV work, discuss current research on arboviral TBVs and present several promising TBV candidates.

A Role for Human Skin Mast Cells in Dengue Virus Infection and Systemic Spread

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious global human disease and mortality. Skin immune cells are an important component of initial DENV infection and systemic spread. Here, we show that mast cells are a target of DENV in human skin and that DENV infection of skin mast cells induces degranulation and alters cytokine and growth factor expression profiles. Importantly, to our knowledge, we also demonstrate for the first time that DENV localizes within secretory granules in infected skin mast cells. In addition, DENV within extracellular granules was infectious in vitro and in vivo, trafficking through lymph to draining lymph nodes in mice. We demonstrate an important role for human skin mast cells in DENV infection and identify a novel mechanism for systemic spread of DENV infection from the initial peripheral mosquito injection site.

Overview of West Nile Virus Transmission and Epidemiology.

West Nile virus (WNV) is a mosquito-borne flavivirus that can cause mild-to-severe disease in humans and horses. WNV was first documented in Uganda in 1937 and passed through the majority of Africa, West Asia, and Europe before arriving in the USA (with infections in New York City in 1999). After the spread of the virus on the US east coast, it traveled westward, northward, and southward through the USA and into Central and South America. WNV can cause fever, rashes, nausea, vomiting, and potentially neuroinvasive disease or death. The virus is sustained through a mosquito-bird-mosquito cycle and there are many species that are competent vectors. Unfortunately, there are no vaccines and the only treatment is supportive care. This chapter highlights the epidemiology and transmission of WNV and provides insight into some of the challenges of controlling WNV disease.

A novel mosquito ubiquitin targets viral envelope protein for degradation and reduces virion production during dengue virus infection

Abstract Background Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant human disease and mortality in the tropics and subtropics. By examining the effects of virus infection on gene expression, and interactions between virus and vector, new targets for prevention of infection and novel treatments may be identified in mosquitoes. We previously performed a microarray analysis of the Aedes aegypti transcriptome during infection with DENV and found that mosquito ubiquitin protein Ub3881 (AAEL003881) was specifically and highly down-regulated. Ubiquitin proteins have multiple functions in insects, including marking proteins for proteasomal degradation, regulating apoptosis and mediating innate immune signaling. Methods We used qRT-PCR to quantify gene expression and infection, and RNAi to reduce Ub3881 expression. Mosquitoes were infected with DENV through blood feeding. We transfected DENV protein expression constructs to examine the effect of Ub3881 on protein degradation. We used site-directed mutagenesis and transfection to determine what amino acids are involved in Ub3881-mediated protein degradation. Immunofluorescence, Co-immunoprecipitation and Western blotting were used to examine protein interactions and co-localization. Results The overexpression of Ub3881, but not related ubiquitin proteins, decreased DENV infection in mosquito cells and live Ae. aegypti. The Ub3881 protein was demonstrated to be involved in DENV envelope protein degradation and reduce the number of infectious virions released. Conclusions We conclude that Ub3881 has several antiviral functions in the mosquito, including specific viral protein degradation. General significance Our data highlights Ub3881 as a target for future DENV prevention strategies in the mosquito transmission vector.

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.

Human C5a Protein Participates in the Mosquito Immune Response Against Dengue Virus.

Abstract Dengue virus (DENV) is transmitted by Aedes spp mosquitoes during a bloodmeal uptake. The bloodmeal consists of host cells, immune factors, and possibly blood-borne pathogens, such as arboviruses. Human cells and immune-related factors, like the complement system, can remain active in the bloodmeal and may be able to interact with pathogens in the mosquito. Previous studies have shown that active complement proteins impact Plasmodium parasite viability in the Anopheles midgut. Thus, we investigated the effects of the human complement on DENV infection in the midgut of Aedes aegypti. Our findings indicate that mosquitoes receiving DENV mixed with normal non-inactivated human serum showed significantly lower viremia than those fed with heat-inactivated serum. This implies that human complement may act to limit DENV infection in the mosquito midgut. In addition, we found that human complement C5a protein was able to directly communicate with mosquito cells, affecting the cell antiviral response against DENV. Our results also show that human C5a protein is able to interact with several membrane-bound mosquito proteins. Together these results suggest an important role of human complement protein in DENV transmission.

Flavivirus Pathogenesis in the Mosquito Transmission Vector

Purpose of ReviewWe hope to provide an overview of flavivirus pathogenesis in the mosquito, including the mosquito immune response to infection.Recent FindingsThere has been a great deal of research in the past decade examining interactions of flaviviruses with mosquito proteins as well as the complex immune reaction to flavivirus infection. We also touch upon novel strategies to prevent mosquito infection with flaviviruses of human importance.SummaryThe mosquito is a crucial component of the flavivirus transmission cycle, and thus, it is vital to thoroughly examine flavivirus pathogenesis in the mosquito vector. There have been some important recent findings in this area yet further research is needed for a comprehensive picture of mosquito-human flavivirus transmission.