Viral Paratransgenesis in the Malaria Vector Anopheles gambiae

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Abstract
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae. Paratransgenesis, the genetic manipulation of mosquito symbiotic microorganisms, is being considered as a potential strategy to control malaria. Microorganisms associated with Anopheles mosquitoes could be manipulated to alter the mosquito's ability to become infected with and transmit the malaria parasites, or reduce mosquito fecundity or lifespan. We identified the first potential microorganism (An. gambiae densovirus; AgDNV) for paratransgenesis of the major malaria vector Anopheles gambiae. AgDNV is highly infectious to An. gambiae larvae, disseminates to adult tissues and is transmitted vertically to subsequent generations. Recombinant AgDNV was able to transduce expression of an exogenous gene (EGFP) in An. gambiae cells and mosquitoes. EGFP-transducing virions infected mosquitoes, expressed EGFP in epidemiologically relevant tissues and were transmitted to offspring in a similar manner to wild-type virus. AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium genes or insect-specific toxins in Anopheles mosquitoes, as well as an easy-to-use system for transient gene expression and RNAi for basic laboratory research.