FIG. 1.

Use of the indirect-immunofluorescence assay to detect the inhibition of DEN replication in mosquito cells and tissues. Cultured mosquito (C6/36) cells were uninfected (A), infected with nonrecombinant dsSIN (TE3′/2J) (B), or infected with recombinant dsSIN expressing the full-length premembrane (prM) protein coding region (567 nt) of the DEN-2 genome in either sense (D2prMs), (C) or antisense (D2prMa) (D) orientation at a MOI of 50. All cells were challenged 48 h later with DEN-2 at a MOI of 0.1. DEN-2 replication was detected 5 days postchallenge by the indirect-immunofluorescence assay. Adult female A. aegypti mosquitoes were injected intrathoracically with DEN-2 alone (E), (magnification ×200) or coinjected with D2prMa (10⁵ TCID₅₀) and DEN-2 (10³ TCID₅₀) (F) (magnification, ×400) and maintained at 28°C for 11 days. Salivary glands were removed and subjected to IFA with the use of anti-DEN E monoclonal primary antibodies and a biotinylated sheep anti-mouse secondary antibody. Fluorescence produced by bound fluorescein-streptavidin (Amersham, Arlington Heights, Ill.) was viewed with an Olympus BH-2 epifluorescence microscope. Cell cultures were counterstained with Evans blue. Cells and tissues positive for DEN-2 E antigen were considered positive for DEN-2 replication.