Molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes

Abstract

Arthropod-borne virus (arbovirus) infections cause a number of emerging and resurgent human and veterinary infectious diseases. Traditional means of controlling arbovirus diseases include vaccination of susceptible vertebrates and mosquito control, but in many cases these have been unavailable or ineffective, and so novel strategies for disease control are needed. One possibility is genetic manipulation of mosquito vectors to render them unable to transmit arboviruses. This review describes recent work to test the concept of pathogen-derived resistance in arthropods by expression of viral genes in mosquito cell cultures and mosquitoes. Sense and antisense genome sequences from La Crosse virus (LAC) (a member of the Bunyaviridae) and dengue viruses serotypes 1 to 4 (DEN-1 to DEN-4) (members of the Flaviviridae) were expressed in mosquito cells from double-subgenomic and replicon vectors based on Sindbis virus (a member of the Togaviridae). The cells were then challenged with homologous or related viruses. For LAC, expression of antisense sequences from the small (S) genome segment, particularly full-length antisense S RNA, effectively interfered with replication of challenge virus, whereas expression of either antisense or sense RNA from the medium (M) segment was completely ineffective in LAC inhibition. Expression of sense and antisense RNA derived from certain regions of the DEN genome also blocked homologous virus replication more effectively than did RNA from other regions. Other parameters of RNA-mediated interference have been defined, such as the time when replication is blocked and the minimum size of effector RNA. The mechanism of RNA inhibition has not been determined, although it resembles double-stranded RNA interference in other nonvertebrate systems. Prospects for application of molecular strategies to control arbovirus diseases are briefly reviewed.

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.