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. 2014 Mar 1;1(1):21-31.
doi: 10.1007/s40475-013-0007-2.

Novel Genetic and Molecular Tools for the Investigation and Control of Dengue Virus Transmission by Mosquitoes

Affiliations

Novel Genetic and Molecular Tools for the Investigation and Control of Dengue Virus Transmission by Mosquitoes

Alexander W E Franz et al. Curr Trop Med Rep. .

Abstract

Aedes aegypti is the principal vector of dengue virus (DENV) throughout the tropical world. This anthropophilic mosquito species needs to be persistently infected with DENV before it can transmit the virus through its saliva to a new vertebrate host. In the mosquito, DENV is confronted with several innate immune pathways, among which RNA interference is considered the most important. The Ae. aegypti genome project opened the doors for advanced molecular studies on pathogen-vector interactions including genetic manipulation of the vector for basic research and vector control purposes. Thus, Ae. aegypti has become the primary model for studying vector competence for arboviruses at the molecular level. Here, we present recent findings regarding DENV-mosquito interactions, emphasizing how innate immune responses modulate DENV infections in Ae. aegypti. We also describe the latest advancements in genetic manipulation of Ae. aegypti and discuss how this technology can be used to investigate vector transmission of DENV at the molecular level and to control transmission of the virus in the field.

Keywords: Aedes aegypti; Aedes albopictus; JAK-STAT; RIDL; RNA interference; TALEN; Toll; Wolbachia; apoptosis; dengue virus; effector gene; gene expression; gene-knockout; homing endonuclease; innate immunity; mosquito; population replacement; promoter; site-specific recombination; transgenesis; transposon; viral tropical medicine; virus transmission; zinc finger nuclease.

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Figures

Fig. 1
Fig. 1
DENV2 is targeted by the innate RNAi pathway in Aedes aegypti and responds to RNAi pathway manipulation in mosquitoes. (a) Intrathoracic injection of dsRNAs derived from sequences of the RNAi pathway genes ago2 and r2d2 into mosquitoes of the Higgs White Eye (HWE) strain triggers RNAi and leads to transient silencing of these genes. In presence of the impaired RNAi pathway, DENV2 titers are significantly increased at 7 days post-infectious bloodmeal (pbm). Controls: (HWE) mosquitoes injected with PBS or dsRNA derived from the β-Gal gene (non-target control). Each data point represents the virus titer of an individual female. (b) Transgene-mediated, constitutive overexpression of a potent RNAi suppressor (FHV-B2) in midgut tissue of PUbB2 mosquitoes impairs the RNAi pathway by inhibiting dsRNA processing. This leads to a significant increase of DENV2 titers in midguts at 7 days pbm in comparison to the HWE control. Each data point represents the virus titer of an individual female midgut. (c) Transgene-mediated expression of an IR effector complementary to the prM-M gene of DENV2 triggers RNAi against the virus in midguts of bloodfed females. Transgenic mosquitoes of line Carb109 are completely refractory to the virus. In contrast, HWE control mosquitoes are highly susceptible to DENV2 at 7 days pbm. DENV2 bloodmeal titers: 106–107 plaque forming units (pfu)/ml. Each data point represents the virus titer of an individual female. Virus titers were assessed by plaque assays in LLC-MK2 monkey kidney cells. Letters a, b next to numbers represent statistically significant groupings (p-value:<0.05; Tukey-Kramer Test).
Fig. 2
Fig. 2
Tools for the genetic manipulation and control of Aedes aegypti. (a) Principle of the ΦC31 site-directed recombination system. With the help of a transposable element (TE) the phage attachment site attp is anchored in the genome of the docking strain. The docking strain is then ‘super-transformed’ with the attB site containing donor plasmid, which also contains the gene-of-interest. Co-injecting the donor and in vitro transcribed ΦC31 integrase into embryos of the docking strain leads to a recombination event between attP and attB. As a consequence, the entire donor plasmid is integrated at the attP site; attP and attB are converted into attL and attR. Using the ΦC31 site-directed recombination system position effects can be avoided because the transgene is integrated at a defined locus. (b) Principle of the Gal4-UAS binary expression system. A transgenic driver line is generated to express the yeast Gal4 activator from a promoter-of-choice. The responder line contains the Gal4 binding site (UAS) and the gene-of-interest (i.e, the EGFP reporter) to be expressed under control of a minimal promoter such as hsp70. Crossing the two transgenic mosquito lines will result in progeny in which the Gal4 protein is binding to the UAS sequences. As a result, the EGFP reporter is expressed from the promoter-of-choice. The advantage of this system is that tissue-specific promoters can be tested with genes-of-interest in various combinations by setting up simple crossings between different driver and responder lines. (c) Principle of the RIDL system for population reduction of Ae. aegypti. The tetracycline repressible system shown here consists of two components. One component encodes the tetracycline-repressible transcriptional activator, tTAV, which is under control of a promoter-of-choice, i.e., a female-specific promoter (to achieve female-specific killing). The other component contains the tTAV binding site tetO7 linked to a pro-apoptotic gene such as michelob x (AeMx), which is under control of a minimal promoter (hsp70). In absence of tetracycline, tTAV will bind to tetO7, resulting in expression of AeMx. Addition of tetracycline as a food supplement will result in binding of tTAV to tetracycline. Tetracycline-bound tTAV shows an altered structure, which is no longer complementary to tetO7 and consequently, AeMx is not expressed. Abbreviations: TE left and right: lest, right terminal inverted-repeats of the transposable element; 3xP3: eye tissue-specific promoter; EGFP, ECFP, DsRed: fluorescent reporters; SV: transcription termination signal of Simian virus 40; Vg: promoter of the Ae. aegypti vitellogenin 1 gene; hsp70: heat-shock promoter 70 of Drosophila; AeMx: michelob x gene of Ae. aegypti.
Fig. 2
Fig. 2
Tools for the genetic manipulation and control of Aedes aegypti. (a) Principle of the ΦC31 site-directed recombination system. With the help of a transposable element (TE) the phage attachment site attp is anchored in the genome of the docking strain. The docking strain is then ‘super-transformed’ with the attB site containing donor plasmid, which also contains the gene-of-interest. Co-injecting the donor and in vitro transcribed ΦC31 integrase into embryos of the docking strain leads to a recombination event between attP and attB. As a consequence, the entire donor plasmid is integrated at the attP site; attP and attB are converted into attL and attR. Using the ΦC31 site-directed recombination system position effects can be avoided because the transgene is integrated at a defined locus. (b) Principle of the Gal4-UAS binary expression system. A transgenic driver line is generated to express the yeast Gal4 activator from a promoter-of-choice. The responder line contains the Gal4 binding site (UAS) and the gene-of-interest (i.e, the EGFP reporter) to be expressed under control of a minimal promoter such as hsp70. Crossing the two transgenic mosquito lines will result in progeny in which the Gal4 protein is binding to the UAS sequences. As a result, the EGFP reporter is expressed from the promoter-of-choice. The advantage of this system is that tissue-specific promoters can be tested with genes-of-interest in various combinations by setting up simple crossings between different driver and responder lines. (c) Principle of the RIDL system for population reduction of Ae. aegypti. The tetracycline repressible system shown here consists of two components. One component encodes the tetracycline-repressible transcriptional activator, tTAV, which is under control of a promoter-of-choice, i.e., a female-specific promoter (to achieve female-specific killing). The other component contains the tTAV binding site tetO7 linked to a pro-apoptotic gene such as michelob x (AeMx), which is under control of a minimal promoter (hsp70). In absence of tetracycline, tTAV will bind to tetO7, resulting in expression of AeMx. Addition of tetracycline as a food supplement will result in binding of tTAV to tetracycline. Tetracycline-bound tTAV shows an altered structure, which is no longer complementary to tetO7 and consequently, AeMx is not expressed. Abbreviations: TE left and right: lest, right terminal inverted-repeats of the transposable element; 3xP3: eye tissue-specific promoter; EGFP, ECFP, DsRed: fluorescent reporters; SV: transcription termination signal of Simian virus 40; Vg: promoter of the Ae. aegypti vitellogenin 1 gene; hsp70: heat-shock promoter 70 of Drosophila; AeMx: michelob x gene of Ae. aegypti.
Fig. 2
Fig. 2
Tools for the genetic manipulation and control of Aedes aegypti. (a) Principle of the ΦC31 site-directed recombination system. With the help of a transposable element (TE) the phage attachment site attp is anchored in the genome of the docking strain. The docking strain is then ‘super-transformed’ with the attB site containing donor plasmid, which also contains the gene-of-interest. Co-injecting the donor and in vitro transcribed ΦC31 integrase into embryos of the docking strain leads to a recombination event between attP and attB. As a consequence, the entire donor plasmid is integrated at the attP site; attP and attB are converted into attL and attR. Using the ΦC31 site-directed recombination system position effects can be avoided because the transgene is integrated at a defined locus. (b) Principle of the Gal4-UAS binary expression system. A transgenic driver line is generated to express the yeast Gal4 activator from a promoter-of-choice. The responder line contains the Gal4 binding site (UAS) and the gene-of-interest (i.e, the EGFP reporter) to be expressed under control of a minimal promoter such as hsp70. Crossing the two transgenic mosquito lines will result in progeny in which the Gal4 protein is binding to the UAS sequences. As a result, the EGFP reporter is expressed from the promoter-of-choice. The advantage of this system is that tissue-specific promoters can be tested with genes-of-interest in various combinations by setting up simple crossings between different driver and responder lines. (c) Principle of the RIDL system for population reduction of Ae. aegypti. The tetracycline repressible system shown here consists of two components. One component encodes the tetracycline-repressible transcriptional activator, tTAV, which is under control of a promoter-of-choice, i.e., a female-specific promoter (to achieve female-specific killing). The other component contains the tTAV binding site tetO7 linked to a pro-apoptotic gene such as michelob x (AeMx), which is under control of a minimal promoter (hsp70). In absence of tetracycline, tTAV will bind to tetO7, resulting in expression of AeMx. Addition of tetracycline as a food supplement will result in binding of tTAV to tetracycline. Tetracycline-bound tTAV shows an altered structure, which is no longer complementary to tetO7 and consequently, AeMx is not expressed. Abbreviations: TE left and right: lest, right terminal inverted-repeats of the transposable element; 3xP3: eye tissue-specific promoter; EGFP, ECFP, DsRed: fluorescent reporters; SV: transcription termination signal of Simian virus 40; Vg: promoter of the Ae. aegypti vitellogenin 1 gene; hsp70: heat-shock promoter 70 of Drosophila; AeMx: michelob x gene of Ae. aegypti.

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