Molecular biology and pathogenesis of Aedes aegypti densovirus

Abstract

Aedes aegypti densovirus (AeDNV) is a parvovirus that efficiently infects and kills Aedes aegypti mosquitoes. AeDNV has potential for use as a biological control agent, or a transducing vector for the introduction of genes of interest into Aedes aegypti mosquitoes. AeDNV has two promoters shown to be functional by reporter gene analysis. Expression from the structural protein gene promoter (pVP) and nonstructural protein gene promoter (p7) was analyzed by deletion and mutation of constructs containing the lacZ reporter gene fused to the VP and NS1 open reading frames. Primer extension was used to map the transcriptional start site of the structural transcript to a consensus initiator sequence (CAGT) 60 nucleotides upstream from the map unit 61 TATAA sequence previously thought to define the promoter. Mutagenesis and deletion analysis showed that both the TATAA sequence at map unit 59.5 and the initiator sequence are required for efficient expression of LacZ-VP fusions from pVP and mutation of the map unit 61 TATAA sequence had little effect. Mutation of the p7 TATAA sequence abolished gene expression, however, in contrast to the pVP promoter, mutation of the consensus initiator sequence only reduced expression of NS1-lacZ fusion proteins by 60%. Temporal expression analysis of the NS1, NS2, and VP fusion proteins showed that all viral proteins are expressed simultaneously. VP protein is expressed to a higher level than the NS proteins and NS2 is expressed to much higher levels than NS1. A mixture of wild-type AeDNV and transducing virus carrying the GFP gene were used to infect Aedes aegypti larvae. The pathogenesis of AeDNV infection was followed by the observation of GFP expression by fluorescence microscopy. The anal papillae were the primary route of infection, with dissemination to the fat bodies taking approximately two days after infection. Older larvae were less susceptible to infection and more likely to survive into adult stages still expressing GFP. Fifty-six percent of infected larvae lost infected anal papillae after they were observed to shrink or occasionally melanize, suggesting an anti-viral immune response. Adult Aedes aegypti were infected with a mixture of wild-type AeDNV and transducing virus by intrathoracic injection. Ninety-Five percent of mosquitoes showed GFP expression 72 hours after infection. Injection of mosquitoes 24 hours after the ingestion of a blood meal provided optimal infection rates in progeny larvae. Efficiency of vertical transmission of virus paralleled the endocytic activity of developing follicles within the ovaries. GFP expression was detectable in 50% of progeny, however PCR detected the presence of wild type AeDNV in 100% of young larvae. However, GFP expression was found to be transient, usually disappearing by 4 days post hatching. PCR detected AeDNV or GFP DNA in only 16% of surviving adults. These results suggest that intrathoracic injection of AeDNV derived vectors may provide an efficient means of introducing genes of interest into mosquitoes.