Sindbis virus induces the production of a novel class of endogenous siRNAs in Aedes aegypti mosquitoes.

Small RNA regulatory pathways are used to control the activity of transposons, regulate gene expression and resist infecting viruses. We examined the biogenesis of mRNA-derived endogenous short-interfering RNAs (endo-siRNAs) in the disease vector mosquito Aedes aegypti. Under standard conditions, mRNA-derived endo-siRNAs were produced from the bidirectional transcription of tail-tail overlapping gene pairs. Upon infection with the alphavirus, Sindbis virus (SINV), another class of mRNA-derived endo-siRNAs was observed. Genes producing SINV-induced endo-siRNAs were not enriched for overlapping partners or nearby genes, but were enriched for transcripts with long 3' untranslated regions. Endo-siRNAs from this class derived uniformly from the entire length of the target transcript, and were found to regulate the transcript levels of the genes from which they were derived. Strand-specific quantitative PCR experiments demonstrated that antisense strands of targeted mRNA genes were produced to exonic, but not intronic regions. Finally, small RNAs mapped to both sense and antisense strands of exon-exon junctions, suggesting double-stranded RNA precursors to SINV-induced endo-siRNAs may be synthesized from mature mRNA templates. These results suggest additional complexity in small RNA pathways and gene regulation in the presence of an infecting virus in disease vector mosquitoes.

Validation of novel promoter sequences derived from two endogenous ubiquitin genes in transgenic Aedes aegypti.

To date, only a limited number of promoter sequences have been described to drive transgene expression in the disease vector Aedes aegypti. We sought to increase this repertoire by characterizing the ability of upstream sequences derived from the Ae. aegypti Ub(L40) and polyubiquitin genes to drive the expression of marker proteins. Both genomic fragments were able to drive robust expression of luciferase in cultured mosquito cells. Following Mos1-transformation, the Ub(L40) promoter drove strong expression of a fluorescent marker in early larvae and in ovaries, while the polyubiquitin promoter drove robust EGFP expression in all stages of development, including constitutive expression throughout the midgut. These promoter fragments provide two new expression profiles for future Ae. aegypti genetic experiments.

Homing endonucleases catalyze double-stranded DNA breaks and somatic transgene excision in Aedes aegypti.

Aedes aegypti is a major vector of arthropod-borne viruses such as yellow fever virus and dengue viruses. Efforts to discern the function of genes involved in important behaviours, such as vector competence and host seeking through reverse genetics, would greatly benefit from the ability to generate targeted gene disruptions. Homing endonucleases are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this report we demonstrate that the homing endonucleases I-PpoI, I-SceI, I-CreI and I-AniI are all able to induce dsDNA breaks in adult female Ae. aegypti chromosomes as well as catalyze the somatic excision of a transgene. These experiments provide evidence that homing endonucleases can be used to manipulate the genome of this important disease vector.

Sindbis virus-induced silencing of dengue viruses in mosquitoes.

Aedes aegypti were injected intrathoracically with double subgenomic Sindbis (dsSIN) viruses with inserted sequences derived from the genome of one or more of the four dengue (DEN) virus serotypes. Mosquitoes were highly resistant to challenge with homologous DEN viruses from which the effector sequences were derived, and resistance to DEN viruses was independent of the orientation of the effector RNA. dsSIN viruses designed to express RNA derived from the premembrane coding region of DEN-2 prevented the accumulation of DEN2 RNA, and C6/36 cells were highly resistant to DEN-2 virus when challenged at 2, 5 or 8 days after the initial dsSIN virus infections, even though the dsSIN-derived RNA had sharply declined at the later time points. Initiation of resistance occurred prior to or within the first 8 h after challenge with DEN-2 virus. We conclude that DEN viruses are inhibited by a mechanism similar to post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) phenomena described in plants and invertebrates, respectively. The potential occurrence of PTGS or RNAi in mosquitoes and mosquito cells suggests new ways of inhibiting the replication of arthropod-borne viruses in mosquito vectors, studying vector-virus interactions, and silencing endogenous mosquito genes.

Molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes.

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.