Antibodies against CRVP379 recognize native protein in Ae. aegypti.
A. CRVP379 antisera binds recombinant protein. An SDS-PAGE gel was run using rCRVP379 and Western blot analysis was done using the CRVP379 antisera. B. Antibodies were made against mosquito CRVP379 and used to bind endogenous protein in the Aag2 cell line. Aag2 cells were infected with DENV and cell fixed 24 hpi for staining. A representative image is shown at 20X. C. CRVP379 antibodies bind endogenous CRVP379 in mosquito midguts. Ae. aegypti MG were dissected and fixed in 4% paraformaldehyde before staining. Both preimmune sera and sera from CRVP-379 injected mice were used for staining (green). Phalloidin594 was used to highlight midgut structure (red). D. Antisera against CRVP379 recognizes the CRVP379 protein in mosquito cells. Aag2 cells were transfected with an expression construct encoding His-tagged CRVP379 protein. Cells were fixed 48h post-transfection and stained with both CRVP379 antisera and antibody against the His tag. Secondary antibodies were used as indicated. E. CRVP379 is increased in both MG and SG of DENV-infected mosquitoes. Mosquitoes were either infected with DENV or mock solution and organs dissected at 1, 2 and 7 dpi. CRVP379 gene expression was analyzed by qRT-PCR analysis and is shown as ng CRVP379 normalized to actin. DENV was used at 105 PFU/mL for infection in mosquitoes. F. CRVP379 antisera binds mosquito cells and tissues. ELISA analysis was done with Aag2 cell lysate, Aedes aegypti salivary gland extract (SGE) and Aedes aegypti extracted saliva. O.D. values at 450nm are presented on the graph.
