Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome

Abstract

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN viruses expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.

Effect of prophenoloxidase expression knockout on the melanization of microfilariae in the mosquito Armigeres subalbatus

“…Data illustrate that haemolymph PO activity was not totally inhibited in the As-pro-PO Ia dsSIN virus-infected mosquitoes, but the residual PO activity was not sufficient to elicit an effective melanization response. Although the precise mechanism by which the dsSIN-based antisense strategy interferes with gene expression is not known, we do know that the efficiency of RNA-mediated interference is dependent on high sequence identity between the effector and target RNAs Gaines et al , 1996;Higgs et al , 1998). We previously demonstrated that at least two POs are present in Ar.…”

Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vector

“…Creating transgenic mosquitoes refractory for disease transmission could provide a needed breakthrough in combating mosquito vectored diseases. RNAs encoding antisense and /or sense sequences to Flavivirus genomes expressed as part of double subgenomic Sindbis virus expression vectors (Higgs et al , 1998;Gaines et al , 1996;Olsen et al , 1996) have already demonstrated a potential for prohibiting the growth and transmission of these viruses.…”

Arthropod-Borne Flaviviruses and RNA Interference

“…Indeed, it has been suggested that RNAi is a key component of the innate immune response of mosquitoes to viral infections (Blair, 2011). In previous sections, we have described that an exogenous trigger of the RNAi pathway in mosquito-cultured cells (Adelman et al, 2001Gaines et al, 1996;Olson et al, 2002) and A. aegypti mosquitoes (Adelman et al, 2001Gaines et al, 1996;Olson et al, 2002) can mediate resistance against flaviviruses. In addition, it has been shown that WNV (Chotkowski et al, 2008) and DENV (Mukherjee & Hanley, 2010) infection (Mukherjee & Hanley, 2010) of Drosophila cell lines induce functional virus-specific siRNAs that promote a protective RNAi response.…”

“…The authors showed that transient or stable expression of long dsRNAs cognate to the DENV-2 genome in A. albopictus (C6/36) cells inhibited subsequent infection of these cells with DENV-2 (Adelman, Blair, Carlson, Beaty, & Olson, 2001;Adelman et al, 2002;Gaines et al, 1996;Olson et al, 2002). Similar results were observed in dsRNA transduced female A. aegypti mosquitoes (Adelman et al, 2001Gaines et al, 1996;Olson et al, 2002). Furthermore, Wu et al found that a synthetic siRNA molecule targeting a sequence in the prM protein-coding region reduced the cytopathic effect in C6/36 cells and increased cell survival more than twofold (Wu et al, 2010).…”