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.

BackgroundDengue virus (DENV) research has historically been hampered by the lack of a susceptible vertebrate transmission model. Recently, there has been progress towards such models using several varieties of knockout mice, particularly those deficient in type I and II interferon receptors. Based on the critical nature of the type I interferon response in limiting DENV infection establishment, we assessed the permissiveness of a mouse strain with a blunted type I interferon response via gene deficiencies in interferon regulatory factors 3 and 7 (IRF3/7 −/− −/−) with regards to DENV transmission success. We investigated the possibility of transmission to the mouse by needle and infectious mosquito, and subsequent transmission back to mosquito from an infected animal during its viremic period.MethodsMice were inoculated subcutaneously with non-mouse adapted DENV-2 strain 1232 and serum was tested for viral load and cytokine production each day. Additionally, mosquitoes were orally challenged with the same DENV-2 strain via artificial membrane feeder, and then allowed to forage or naïve mice. Subsequently, we determined acquisition potential by allowing naïve mosquitoes on forage on exposed mice during their viremic period.ResultsBoth needle inoculation and infectious mosquito bite(s) resulted in 100% infection. Significant differences between these groups in viremia on the two days leading to peak viremia were observed, though no significant difference in cytokine production was seen. Through our determination of transmission and acquisition potentials, the transmission cycle (mouse-to mosquito-to mouse) was completed. We confirmed that the IRF3/7 −/− −/− mouse supports DENV replication and is competent for transmission experiments, with the ability to use a non-mouse adapted DENV-2 strain. A significant finding of this study was that this IRF3/7 −/− −/− mouse strain was able to be infected by and transmit virus to mosquitoes, thus providing means to replicate the natural transmission cycle of DENV.ConclusionAs there is currently no approved vaccine for DENV, public health monitoring and a greater understanding of transmission dynamics leading to outbreak events are critical. The further characterization of DENV using this model will expand knowledge of key entomological, virological and immunological components of infection establishment and transmission events.

Abstract. Dengue virus (DENV) is the most important mosquito-transmitted flavivirus that is transmitted throughout the tropical and subtropical regions of the world. The primary mosquito vector of DENV in urban locations is Aedes aegypti. Key to understanding the transmission of DENV is the relationship between pathogen and vector. Accordingly, we report our preliminary characterization of the differentially expressed proteins from Ae. aegypti mosquitoes after DENV infection. We investigated the virus-vector interaction through changes in the proteome of the salivary glands of mosquitoes with disseminated DENV serotype 2 (DENV-2) infections using two-dimensional gel electrophoresis and identification by mass spectrometry. Our findings indicate that DENV-2 infection in the Ae. aegypti salivary gland alters the expression of structural, secreted, and metabolic proteins. These changes in the salivary gland proteome highlight the virally influenced environment caused by a DENV-2 infection and warrant additional investigation to determine if these differences extend to the expectorated saliva.

Dengue virus (DENV), the etiologic agent of dengue fever, is transmitted during probing of human skin by infected-mosquito bite. The expectorated viral inoculum also contains an assortment of mosquito salivary proteins that have been shown to modulate host hemostasis and innate immune responses. To examine the potential role of mosquito probing in DENV establishment within the vertebrate host, we inoculated mice intradermally with DENV serotype 2 strain 1232 at sites where Aedes aegypti had or had not probed immediately prior. We assayed these sites 3 h postinoculation with transcript arrays for the Toll-like receptor (TLR), RIG-I-like receptor, and NOD-like receptor signaling pathways of the innate immune system. We then chose TLR7, transcription factor p65 (RelA), gamma interferon (IFN-␥), and IFN-␥-inducible protein 10 (IP-10) from the arrays for further investigation and assayed these transcripts at 10 min, 3 h, and 6 h postinoculation. The transcripts for TLR7, RelA, IFN-␥, and IP-10 were significantly downregulated between 2-and 3-fold in the group subjected to mosquito probing relative to the virusonly inoculation group at 3 h postinoculation. A reduction in these transcripts could indicate reduced DENV recognition and antigen presentation and diminished inhibition of viral replication and spread. Further, mosquito probing resulted in viremia titers significantly higher than those in mice that did not receive probing. A. aegypti probing has a significant effect on the innate immune response to DENV infection and generates an early immune environment more permissive to the establishment of infection. Dengue virus (DENV), the etiologic agent of dengue fever, is an enveloped, positive-sense, single-stranded RNA virus of the genus Flavivirus. DENV prevalence has increased in recent decades and now infects an estimated 50 to 100 million humans worldwide annually of approximately 2.5 billion at risk of infection (1). The increased risk of infection can be attributed, in part, to the continued growth and urbanization of human populations and an expanded global distribution of the primary vectors of DENV, the mosquitoes Aedes aegypti and A. albopictus (2). Feeding by these vectors on vertebrate hosts is initiated by probing that results in physical damage to the epithelium and vasculature, as well as the simultaneous introduction of virus and saliva into host tissues (3-6). This saliva contains many pharmacologically important proteins that modulate host hemostasis and innate immune responses, which in turn facilitate blood feeding and virus transmission (7-9). As exogenous antigens, both the salivary proteins and the virus encounter the vertebrate host immune system and consequently could have an effect on the environment of the bite site during viral establishment.To investigate the impact of the vector on mosquito-borne viral infection, researchers have analyzed viral titers and/or immunological response markers by using various methods of mosquito involvement, including salivary gland extract (SGE) (10-16), i...

Dengue virus (DENV) is transmitted in the saliva of the mosquito vector Aedes aegypti during blood meal acquisition. This saliva is composed of numerous proteins with the capacity to disrupt hemostasis or modulate the vertebrate immune response. One such protein, termed “aegyptin,” is an allergen and inhibitor of clot formation, and has been found in decreased abundance in the saliva of DENV-infected mosquitoes. To examine the influence of aegyptin on DENV infection of the vertebrate, we inoculated IRF-3/7 −/− −/− mice with DENV serotype 2 strain 1232 with and without co-inoculation of aegyptin. Mice that received aegyptin exhibited decreased DENV titers in inoculation sites and in circulation, as well as increased concentrations of GM-CSF, IFN-γ, IL-5, and IL-6, at 48 hours post inoculation when compared to mice that received inoculation of DENV alone. These and other data suggest that aegyptin impacts DENV perpetuation via elevated induction of the immune response.