Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

Abstract

BackgroundArthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.ResultsB2 protein expression from SINV (TE/3'2J) inhibited the accumulation of non-specific small RNAs in Aedes aegypti mosquito cell culture and virus-specific small RNAs both in infected cell culture and Ae. aegypti mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2) compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in Ae. aegypti.ConclusionWe show that RNAi is active in Ae. aegypti cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.

Making connections in insect innate immunity

“…The immune response modulates outcomes of infection such as viral load, incubation period required for transmission, and viral pathogenesis in the vector (1,2). As insects appear to lack an adaptive immune response characteristic of vertebrates, this controlling response occurs through activation of apparently simple, linear innate immunity pathways.…”

Learning from the Enemy: Innate RNA Interference Renders Mosquitos Asymptomatic to Arboviral Infection and Provides Researchers with New Approaches to Subdue its Effects

“…Resulting from endogenous transcripts being folded into hairpin like structures, they are loaded into Argonaute proteins in order to guide the complex to its target [15]. At most 7 bases of miRNA need to be matched to the target for translational repression to occur.…”

“…While only a portion of the surveyed research research [11,12,15] studied these pathways in mosquitoes, the conservation of these mechanisms across species indicate that many of the same molecules and mechanisms can be studied to understand the human immune reaction to arboviruses. Researchers should be aware that RNA interference provides them with the tools to interfere and manipulate how arboviruses interacts with cellular mechanisms.…”

Antiviral responses of arthropod vectors: an update on recent advances

“…One interesting observation is that while some arboviruses do have RNAi evasion strategies [111], they generally do not encode efficient suppressors of RNAi. Strong suppression of the antiviral RNAi response could be detrimental to vector survival [24,84] and thus to the transmission of the arbovirus. The only arboviruses known to encode suppressors of RNAi are flaviviruses.…”