Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus

Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain

Evaluación preliminar de la estrategia de información, comunicación y educación para el control del aedes aegypti, en la dorada, colombia

Objetivo Estimar el impacto de la estrategia de Información, Educación y Comunicación sobre la infestación de Aedes aegypti en viviendas de La Dorada. Métodos Estudio transversal adelantado entre junio y julio de 2008 en La Dorada, Colombia. Se seleccionaron 228 viviendas donde se midió presencia del vector a través del método rapid sweeping para formas inmaduras en tanques y de captura activa para adultos. Simultáneamente, una encuesta midió la exposición a la estrategia. La base de datos se analizó en EPI info 2000. Se calcularon Razones de Prevalencia e intervalos de confianza al 95 %. Resultados Las viviendas que no tenían larvas se caracterizaban por tener mujeres cabeza de familia o moradores con conocimiento acerca de las larvas y la forma de transmisión del dengue. Aquellas viviendas donde se lavaba el tanque de agua al menos una vez por semana, carecían de formas inmaduras. La presencia de mosquitos adultos era más frecuente en las casas que tenían más de un tanque. Cerca del 80 % de los encuestados adquirieron el conocimiento de transmisión del dengue a través de la estrategia. Conclusiones Si bien la estrategia no ha sido aplicada en todos sus componentes, se encontraron factores que podrían tener un efecto protectivo al estar relacionados con ausencia del vector. El estudio revela solo un éxito parcial de la estrategia en La Dorada.Objective Estimating the impact of the Information, Education and Communication strategy on Aedes aegypti infestation in homes in La Dorada, Colombia. Methods A cross-sectional study was carried out during June and July 2008 in LaDorada; 228 houses were chosen and vector presence was measured in two ways: immature forms were captured by the rapid sweeping method in water tanks and adults were captured by active collection. A survey simultaneously measuredexposure to the strategy. EPI Info 2000 was used for analysing the database so created. Prevalence ratios and 95 % confidence intervals were calculated. Results A relationship was found between being a housewife and not having larvaepresent in the home. No immature forms were found in houses where somebody washed the water tank at least once a week. There were also no larvae in homes where people had knowledge about larvae and dengue transmission. Thepresence of adult forms was found in houses having more than one water tank. Almost 80 % of the interviewees acquired knowledge about dengue transmission through the strategy.Conclusions Although all strategy components had not been implemented, several factors were found which might have a protective effect (i.e. being related to vector absence). The study showed that the strategy achieved only partial successin La Dorada

Effects of RNAi knockdown of Aedronc, Caspase-16 or Nautilus on the development of DENv in the refractory Cali-MIB strain of <i>Ae. aegypti</i>.

<p>The expected proportions in the Cali-MIB colony (53% Susceptible: 47% Resistant) were maintained in naïve and Nautilus injected insects. Knockdown of Caspase-16 and Aedronc using RNAi increased the proportion of susceptible mosquitoes to 62% in Caspase 16 Kd insects (Chi² =  = 0.39; p = 0.53) and 78.6% in the Aedronc Kd insects (Chi² = 3.9; p = 0.03). The numbers in brackets above each pair of bars indicates the # of mosquitoes in 3–5 replicates on which these summaries are based. The * indicates significant difference between Aedronc knockdown and naïve and Nautilus injected controls.</p

Within Strain Comparisons: Relative expression of Argonaute-2, AeIAP1, caspase-16, Aedronc and Aedredd in the midguts of <i>Aedes aegypti</i> in strains that are Refractory (Cali-MIB: Left Panel) or Susceptible (Cali-S: Right Panel) to Dengue virus in the presence (black bars) or absence (white bars) of Dengue virus-2 in the bloodmeal.

<p>The expression levels in the bloodfed samples were arbitrarily set at 1 and the expression levels in the presence of the virus represent fold-differences from the susceptible controls. The bars were geometrically adjusted (2^−ΔCT)+SD.-2^−ΔCT.</p

Between Strain Comparisons: Differential expression of Argonaute-2, AeIAP1, Caspase-16, Aedronc, and Aedredd in the midguts of Resistant Cali-MIB (black bars) and Susceptible Cali-S mosquitoes (white bars) at the same timepoints after exposure to a bloodmeal containing DENv-2 (top panel) or blood alone (bottom panel).

<p>In each pairwise comparison the expression level in the Cali-S strain was arbitrarily set at 1 and the expression levels in Cali-MIB strain represents fold-differences in expression within that pair. The bars are geometric adjusted (2^−ΔCT)+SD.</p

Gene expression ratios comparing midgut gene expression of Argonaute-2, AeIAP1, Caspase-16, Aedronc, and Aedredd over time, compared with time zero (ΔCt), in Cali-S and Cali-MIB strains exposed to blood or blood+DENv-2, as well as between-strain ratios.

<p>Within strains, the treatments blood and blood+DENv-2 are also compared. ΔCt values were estimated from the multiple regression analysis and the expression ratios were estimated by exponentiating (2^−ΔCT).</p>a<p>Expression ratio 1 as this is the reference category.</p>b<p>Blood vs Blood+DENv-2.</p

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission