Dataset Information

Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

ABSTRACT: We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the development of a non-defective recombinant Aedes aegypti densovirus (AaeDV) miRNA expression system, mediated by an artificial intron, using an intronic miRNA expression strategy. We demonstrated that this recombinant vector could be used to overexpress endogenous miRNAs or to decrease endogenous miRNAs by generating antisense sponges to explore the biological functions of miRNAs. In addition, the vector could express antisense-miRNAs to induce efficient gene silencing in vivo and in vitro. The recombinant virus effectively self-replicated and retained its secondary transmission ability, similar to the wild-type virus. The recombinant virus was also genetically stable. This study demonstrated the first construction of a non-defective recombinant MDV miRNA expression system, which represents a tool for the functional analysis of mosquito genes and lays the foundation for the application of viral paratransgenesis for dengue virus control.

SUBMITTER: Liu P

PROVIDER: S-EPMC4754678 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

Liu Peiwen P Li Xiaocong X Gu Jinbao J Dong Yunqiao Y Liu Yan Y Santhosh Puthiyakunnon P Chen Xiaoguang X

Scientific reports 20160216

We previously reported that mosquito densoviruses (MDVs) are potential vectors for delivering foreign nucleic acids into mosquito cells. However, considering existing expression strategies, recombinant viruses would inevitably become replication-defective viruses and lose their ability for secondary transmission. The packaging limitations of the virion represent a barrier for the development of MDVs for viral paratransgenesis or as high-efficiency bioinsecticides. Herein, we report the developme ...[more]

PMID: 26879823

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Project description:BackgroundIn eukaryotic organisms, Piwi-interacting RNAs (piRNAs) control the activities of mobile genetic elements and ensure genome maintenance. Recent evidence indicates that piRNAs are involved in multiple biological pathways, including transcriptional regulation of protein-coding genes, sex determination and even interactions between host and pathogens. Aedes albopictus is a major invasive species that transmits a number of viral diseases in humans. Ae. albopictus has the largest genome and the highest abundance of repetitive sequences when compared with members that belong to Culicidae with a published genome. Analysis of piRNA profiles will provide a developmental and evolutionary perspective on piRNAs in Ae. albopictus.MethodspiRNAs were identified and characterized during the development of Ae. albopictus, and piRNA expression patterns in adult males and females as well as sugar-fed females and blood-fed females were compared.ResultsOur results reveal that, despite the large genome size of Ae. albopictus, the piRNA pool of Ae. albopictus (1.2 × 107) is smaller than those of Aedes aegypti (1.7 × 107) and Drosophila melanogaster (1.6 × 107). In Ae. albopictus, piRNAs displayed the highest abundance at the embryo stage and the lowest abundance at the pupal stage. Approximately 50 % of the piRNAs mapped to intergenic regions with no known functions. Approximately 30 % of the piRNAs mapped to repetitive elements, and 77.69 % of these repeat-derived piRNAs mapped to Class I TEs; 45.42 % of the observed piRNA reads originated from piRNA clusters, and most of the top 10 highest expressed piRNA clusters and 100 highest expressed piRNAs from each stage displayed biased expression patterns across the developmental stages. All anti-sense-derived piRNAs displayed a preference for uridine at the 5' end; however, the sense-derived piRNAs showed adenine bias at the tenth nucleotide position and a typical ping-pong signature, suggesting that the biogenesis of piRNAs was conserved throughout development. Our results also show that 962 piRNAs displayed sex-biased expression, and 522 piRNAs showed higher expression in the blood-fed females than in the sugar-fed females.ConclusionsOur results suggest that piRNAs, aside from silencing transposable elements in Ae. albopictus, may have a role in other biological pathways.

Project description:BackgroundMicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression in a variety of organisms, including insects, vertebrates, and plants. miRNAs play important roles in cell development and differentiation as well as in the cellular response to stress and infection. To date, there are limited reports of miRNA identification in mosquitoes, insects that act as essential vectors for the transmission of many human pathogens, including flaviviruses. West Nile virus (WNV) and dengue virus, members of the Flaviviridae family, are primarily transmitted by Aedes and Culex mosquitoes. Using high-throughput deep sequencing, we examined the miRNA repertoire in Ae. albopictus cells and Cx. quinquefasciatus mosquitoes.ResultsWe identified a total of 65 miRNAs in the Ae. albopictus C7/10 cell line and 77 miRNAs in Cx. quinquefasciatus mosquitoes, the majority of which are conserved in other insects such as Drosophila melanogaster and Anopheles gambiae. The most highly expressed miRNA in both mosquito species was miR-184, a miRNA conserved from insects to vertebrates. Several previously reported Anopheles miRNAs, including miR-1890 and miR-1891, were also found in Culex and Aedes, and appear to be restricted to mosquitoes. We identified seven novel miRNAs, arising from nine different precursors, in C7/10 cells and Cx. quinquefasciatus mosquitoes, two of which have predicted orthologs in An. gambiae. Several of these novel miRNAs reside within a ~350 nt long cluster present in both Aedes and Culex. miRNA expression was confirmed by primer extension analysis. To determine whether flavivirus infection affects miRNA expression, we infected female Culex mosquitoes with WNV. Two miRNAs, miR-92 and miR-989, showed significant changes in expression levels following WNV infection.ConclusionsAedes and Culex mosquitoes are important flavivirus vectors. Recent advances in both mosquito genomics and high-throughput sequencing technologies enabled us to interrogate the miRNA profile in these two species. Here, we provide evidence for over 60 conserved and seven novel mosquito miRNAs, expanding upon our current understanding of insect miRNAs. Undoubtedly, some of the miRNAs identified will have roles not only in mosquito development, but also in mediating viral infection in the mosquito host.

Dataset Information

Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

Publications

Development of non-defective recombinant densovirus vectors for microRNA delivery in the invasive vector mosquito, Aedes albopictus.

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