Transduction ofAedes aegyptiMosquitoes with Vectors Derived fromAedesDensovirus

Abstract

Aedes densovirus (AeDNV)-based constructs that express green fluorescent protein (GFP) from either the P7 or the P61 promoter were made. The construct in which GFP protein was expressed as a fusion protein to the C-terminus of NS1 (NS1-GFP) showed the highest level of GFP expression. This hybrid NS1-GFP protein preserved the biological functions of the parental proteins: it showed GFP fluorescence, it stimulated expression from the virus promoters, and it facilitated rescue and replication of the cloned AeDNV genome. Similar to NS1, the hybrid NS1-GFP localized in the nucleus predominantly in a punctate pattern. Transducing virus particles carrying the NS1-GFP gene infected mosquito larvae. Expression of GFP was detected as early as 48 h postinfection and in larval and pupal stages. Midgut, hindgut, and Malpighian tubule cells expressed GFP soon after transduction. However, the anal papillae were the most commonly infected organ system. The anal papillae are syncytia and regulate ion concentration in the hemolymph of mosquito larvae, and they might be a novel route of mosquito larvae infection with densoviruses.

Junonia coenia Densovirus-Based Vectors for Stable Transgene Expression in Sf9 Cells: Influence of the Densovirus Sequences on Genomic Integration

“…The cloning of the entire JcDNV genome into pBR322 led to a recombinant construct (pBRJ) that retained the capacity to produce infectious particles when injected into larvae of the sensitive host Spodoptera littoralis or transfected into lepidopteran cell lines (20,24,25). The availability of plasmids carrying an infectious sequence of either the JcDNV or the Aedes aegypti densovirus genome has prompted the study of densoviruses as expression vectors (1,2,5,10,16,31). The A. aegypti densovirus, a prototype of the Brevidensovirus genus, has been developed as a gene transfer vehicle that is able to transduce genes into mosquito larvae by typical routes of in-fection, opening the potential for gene introduction into natural populations (1,2).…”

A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus

“…The p7 promoter was slightly more efficient than p61. 17,18,26 Therefore, in this study, we used different expression strategies for gene of the BmK IT1 toxin and evaluated which was the best for producing a high level of expression, and preserved wt viral function. For high levels of expression and packaging, all expression constructs require the preservation of the entire NS1 gene and a suitable length of the genome, as described previously.…”

“…In p61NTS-BIT-GFP, the BmK IT1 gene was cloned into the VP ORF and transcribed from the p61 promoter, and 77 aa of VP1 that contained a putative NTS sequence was fused to the N-terminal instead; it has been confirmed previously that an additional NTS can target the protein into the nucleus of mosquito cells and increase the expression level of protein. 17,18,27 From a safety standpoint, it is important to track the genetic stability of recombinant densovirus and their presence in vitro and in vivo . For this purpose, GFP was expressed constitutively as a visible marker in p61NTS-BIT-GFP and p7NS1-BIT-GFP.…”

“…The GFP fusion protein was detected throughout the cell but mainly in nuclei where it showed a punctate or uniform pattern of nuclear distribution ( Figure 2 ); this was consistent with the pattern of p7NS1-GFP expression reported previously. 18 This distribution indicates that functions that are vital to the intracellular localization of NS1 or NTS are preserved in the recombinant plasmid, which was an important premise to ensure a high level of expression of the target gene. We identified the expression of BmK IT1 by RT-PCR and immunoblotting with the use of gene-specific primers and anti-BmK IT1 polyclonal immunoglobulin G (IgG), respectively.…”

Molecular Strategies for Interrupting Arthropod-Borne Virus Transmission by Mosquitoes

“…Insect parvoviruses of the genus Densovirus also are potential gene transfer vectors for mosquitoes (2). The Aedes densonucleosis virus is being developed and examined by Jonathan Carlson and colleagues (3,23) at AIDL.…”