Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

Summary

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

Abstract

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.

Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.

This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1^st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

Introduction

Mosquito-borne diseases such as malaria, dengue fever, zika fever, and yellow fever, are major international public health problems that continue to account for a significant fraction of the global infectious disease burden^1,2. Conventional insecticides, which have been used in response to vectors, are a major component of sustainable integrated mosquito control strategy for the prevention of mosquito-borne diseases. However, such strategies have proven to be relatively ineffective or undesirable due to the associated negative environmental impacts as well as the evolution of resistance in mosquito populations....

Protocol

All protocols were approved by the Ethical Committee of Southern Medical University.

1. Designing an Artificial Intron

NOTE: The artificial intron used in this work is 65 bp in length, and the sequence is 5'-GTAAGAGTCGATCGACGCGTTACTAACTGGTACCTCTTCTTTTTTTTTTGATATCCTGCAG-3'.

1. Place the HpaI restriction site on both ends of artificial intron so that HpaI digestion can release the intron from plasmids (see Figure 1A).
2. Place the two XbaI restriction sites in the intron so that XbaI digestion can be used to i....

Results

Strategies for rAaeDV construction 
A defective rAaeDV vector was generated to express the DsRed gene in mosquito larvae. The resulting plasmid contained a NS1-DsRed fusion protein cassette with the VP protein deleted (Figure 1A). rAaeDV plasmids containing expression constructs for miRNA, miRNA sponge, shRNA and amiRNA were designed (shown in Figure 1B). For example, a rAaeD.......

Discussion

It is important to overcome two of the key barriers that limit rAaeDV construction. The first is the production of defective recombinant virus. It has been reported that MDV can be used as a vector to express appropriately sized foreign genes, such as scorpion insect-specific neurotoxins20 and the GFP protein; however, regardless of the construction strategy, once the ORFs of MDV are inserted or replaced with exogenous genes, virions cannot form independently unless a helper plasmid is cotransfected to supply the missing.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge machine	Thermo Scientific	75004260
Centrifuge System	Beckman Coulter	363118
GeneJET Gel Extraction Kit	Thermo Scientific	K0691
E.Z.N.A. Plasmid Midi Kit	Omega Biotech	D6904
Purified Agar	OXOID	LP0028A
FastAP Thermosensitive Alkaline Phosphatase (1 U/µL)	Thermo Scientific	EF0651
FastDigest HpaI	Thermo Scientific	FD1034
FastDigest XbaI	Thermo Scientific	FD0684
T4 DNA Ligase (5 U/µL)	Thermo Scientific	EL0011
TransStbl3 Chemically Competent Cell	Beijing Transgen Biotech	CD521-01
Ampicillin	Beijing Tiangen Biotech	RT501
Proteinase K	Promega	MC5008
SuperReal PreMix Plus (SYBR Green)	Beijing Tiangen Biotech	FP205