Abstract

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.

Highlights

  • Dengue viruses (DENv), transmitted to humans by infected mosquitoes, cause an estimated 50–100 million cases of Dengue fever (DF),500,000 cases of Dengue Hemorrhagic Fever, and .20,000 deaths per year [1,2]

  • Insects recognize unique pathogenassociated molecular patterns (PAMPs) [11], using pattern recognition receptors (PRRs) [12], and activate response pathways such as the IMD and Toll pathways [13] which lead to elimination of parasites through phagocytosis, proteolytic cascades, and synthesis of potent antimicrobial peptides (AMPs) [14,15]

  • The titer of the Dengue-2 virus (DENv-2) preparation used was monitored throughout all selections and ranged from 108 to 108.5 TCID50/mL at the beginning of virus exposure and from 107.2 to 107.4 TCID50/mL at the end of the exposure period

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Summary

Introduction

Dengue viruses (DENv), transmitted to humans by infected mosquitoes, cause an estimated 50–100 million cases of Dengue fever (DF), ,500,000 cases of Dengue Hemorrhagic Fever, and .20,000 deaths per year [1,2]. There is no available vaccine or effective treatment for DENv. Given the limited success achieved through classical vector control [4], many new strategies to reduce transmission have been proposed including the use of genetically modified vectors [5,6,7] or the use of natural symbionts such as Wolbachia [8,9,10]. Given the limited success achieved through classical vector control [4], many new strategies to reduce transmission have been proposed including the use of genetically modified vectors [5,6,7] or the use of natural symbionts such as Wolbachia [8,9,10] The development of such strategies requires extensive knowledge of the molecular interactions between virus and vector and how these determine vector competence (VC), the intrinsic ability of an arthropod to transmit a pathogen. The VC of Ae. aegypti has been studied extensively through the selection of strains with different susceptibilities [18,26,27,28,29,30] but no specific genes have been identified as determinants of DENv susceptibility and it is unknown if all geographic strains of Ae

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