Defective Interfering Particles (DIPs) are naturally occurring viral particles of RNA viruses that contain RNA genomes with large internal deletions (90-99% of parent virus genome size). DIPs are non-pathogenic, only replicate in the presence of full-genome virus and inhibit parent virus replication. DIPs are therefore being developed as novel therapeutics against RNA viruses, including Influenza. DIP genomes from dengue virus (DENV) have been isolated from dengue patients but not previously from mosquitoes. The role of mosquitoes in the generation or transmission of DIPs is currently unknown.
We investigated the occurrence of DIP genomes (referred to as Defective interfering RNAs, or DI RNAs) in DENV infected mosquitoes and tested the virus suppression ability of a naturally occurring DENV-2 DI RNA. We tested whether DI RNAs accumulate during DENV infection by orally challenging mosquitoes with DENV via glass membrane feeders, incubating these for 14 days and detecting DIP RNAs from bodies, legs and wings and saliva by RT-PCR, cloning and sequencing. We then tested whether the DI RNA from a therapeutic DIP candidate was capable of inhibiting parent virus replication, by challenging mosquitoes with DENV-2 and microinjecting mosquitoes with 1 µg of the DI RNA at 2 d post infection.
We show that DIPs accumulate within mosquito tissues and form large populations with a high diversity of RNA sequences (30-58 variants identified per serotype for DENV-2 and DENV-3), including variants previously isolated from patient sera. We isolated DIPs from mosquito saliva, demonstrating that DIPs can be transmitted to new hosts, and show significant reduction in DENV-2 titre in mosquitoes following microinjection with a DI RNA molecule.
Defective Interfering Particles are natural components of DENV infection in mosquitoes, however, particular DIPs are inhibitory at higher concentrations and have potential to be novel therapeutic agents.