A dengue receptor as possible genetic marker of vector competence in Aedes aegypti

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.

(1) We determined the MG proteins that bind DENV by an overlay protein binding assay (VOPBA) in Ae. aegypti mosquitoes of the DS3, DMEB and IBO-11 strains. The main protein identified had an apparent molecular weight of 67 kDa, although the protein identified in the IBO-11 strain showed a lower mass (64 kDa). (2) The midgut proteins recognized by DENV were also determined by VOPBA after two-dimensional gel electrophoresis. (3) To determine whether the same proteins were identified in all three strains, we obtained polyclonal antibodies against R67 and R64 and tested them against the three strains by immunoblotting; both antibodies recognized the 67 and 64 kDa proteins, corroborating the VOPBA results. (4) Specific antibodies against both proteins were used for immunofluorescent location by confocal microscopy; the antibodies recognized the basal lamina all along the MG, and cell membranes and intercellular spaces from the middle to the end of the posterior midgut (pPMG) in the neighborhood of the hindgut. (5) Quantitative analysis showed more intense fluorescence in DS3 and DMEB than in IBO-11. (6) The viral envelope antigen was not homogeneously distributed during MG infection but correlated with MG density and the distribution of R67/R64.

In this paper we provide evidence that the 67 kDa protein (R67/R64), described previously as a DENV receptor, is related to vector competence in Ae. aegypti. Consequently, our results strongly suggest that this protein may be a marker of vector competence for DENV in Ae. aegypti mosquitoes.