The dengue virus NS1 protein alters <i>Aedes aegypti</i> midgut permeability and favors virus dissemination

The dengue virus is an arbovirus of public health importance transmitted by <i>Aedes aegypti</i> mosquitoes. The NS1 protein is a multifunctional glycoprotein, highly conserved among Orthoflaviviruses, that is secreted from infected cells, circulates in the sera of dengue patients at high concentrations, and has been involved with pathogenesis by several mechanisms, including vascular leakage and immune evasion. However, the role of NS1 within the mosquito vector is not fully understood. In this study, we observed that feeding female <i>Aedes aegypti</i> mosquitoes with blood meals supplemented with recombinant NS1 resulted in increased midgut permeability, as evaluated by the diffusion of the non-toxic dye Brilliant Blue FCF into the hemocoel. Histological and transmission electron microscopy analyses revealed epithelial damage and disruption of the midgut cellular junctions. Immunofluorescence assays further demonstrated the delocalization of septate junction proteins essential for epithelial integrity. In addition, metalloproteinase expression in the midgut was also reduced. All these effects were abolished when NS1 was heat-inactivated, indicating that they were NS1-dependent. Significantly, infected blood meals resulted in enhanced dissemination of DENV to secondary organs and earlier virus presence in the mosquito salivary glands, in comparison with meals treated with specific NS1 antibodies. Our findings reveal a novel function for NS1 in mosquitoes and expand the understanding of NS1 functions beyond its human pathophysiological role. In addition, our findings highlight NS1 as a strategic intervention point to reduce transmission efficiency in mosquitoes.IMPORTANCEDengue is the most important viral disease transmitted by mosquitoes to humans. Thus, a deeper understanding of the virus-vector interaction is required for the development of control measures and the mosquito transmission capacity. In this work, we present evidence indicating that the NS1 protein plays a role in the establishment and dissemination of the dengue virus infection in <i>Aedes aegypti</i> mosquitoes. Ingested NS1 was found to disrupt the septate junctions and to inhibit metalloproteinase expression of the midgut, allowing the virus to escape the digestive tract. In addition, NS1 was found to promote virus dissemination into the hemocoel, carcass, and salivary glands. These findings uncover new functions for NS1 in the mosquito and highlight ways to interfere with vector competence.