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Viral paratransgenesis in the malaria vector Anopheles gambiae.

Ren X, Hoiczyk E, Rasgon JL - PLoS Pathog. (2008)

Bottom Line: To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes.Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: The Johns Hopkins Malaria Research Institute, Baltimore, Maryland, United States of America.

ABSTRACT
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

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IFA detection of AgDNV infection of dissected adult tissues.Mosquitoes were infected as first-instar larvae with AgDNV purified from Sua5B cells. A, B: midgut; C, D: ovary. A, C: infected with AgDNV; B, D: control. Blue: cell nuclei stained with DAPI, green: densovirus.
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ppat-1000135-g002: IFA detection of AgDNV infection of dissected adult tissues.Mosquitoes were infected as first-instar larvae with AgDNV purified from Sua5B cells. A, B: midgut; C, D: ovary. A, C: infected with AgDNV; B, D: control. Blue: cell nuclei stained with DAPI, green: densovirus.

Mentions: To test for transtadial transmission and dissemination of AgDNV in adult mosquitoes, we infected first-instar An. gambiae larvae, transferring them to clean virus-free water after 2 days. Uninfected control larvae were exposed to culture media. After adult emergence, we dissected adult tissues and performed densovirus-specific immunofluorescence microscopy. AgDNV clearly disseminates and infects adult midgut and ovary (Figure 2). We then assessed whether AgDNV could be transmitted to subsequent generations. We treated mosquitoes for 24 hours as larvae with AgDNV, which were reared to adulthood, bloodfed, allowed to oviposit, and their offspring reared to adulthood and assayed for AgDNV by PCR. Fifty percent of treated mosquitoes were positive for virus by PCR (N = 42). Twenty-eight percent (N = 71) of their offspring were positive for infection, indicating that AgDNV was transmitted between generations, either by vertical transmission or by horizontal transmission from adults to larvae.


Viral paratransgenesis in the malaria vector Anopheles gambiae.

Ren X, Hoiczyk E, Rasgon JL - PLoS Pathog. (2008)

IFA detection of AgDNV infection of dissected adult tissues.Mosquitoes were infected as first-instar larvae with AgDNV purified from Sua5B cells. A, B: midgut; C, D: ovary. A, C: infected with AgDNV; B, D: control. Blue: cell nuclei stained with DAPI, green: densovirus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2500179&req=5

ppat-1000135-g002: IFA detection of AgDNV infection of dissected adult tissues.Mosquitoes were infected as first-instar larvae with AgDNV purified from Sua5B cells. A, B: midgut; C, D: ovary. A, C: infected with AgDNV; B, D: control. Blue: cell nuclei stained with DAPI, green: densovirus.
Mentions: To test for transtadial transmission and dissemination of AgDNV in adult mosquitoes, we infected first-instar An. gambiae larvae, transferring them to clean virus-free water after 2 days. Uninfected control larvae were exposed to culture media. After adult emergence, we dissected adult tissues and performed densovirus-specific immunofluorescence microscopy. AgDNV clearly disseminates and infects adult midgut and ovary (Figure 2). We then assessed whether AgDNV could be transmitted to subsequent generations. We treated mosquitoes for 24 hours as larvae with AgDNV, which were reared to adulthood, bloodfed, allowed to oviposit, and their offspring reared to adulthood and assayed for AgDNV by PCR. Fifty percent of treated mosquitoes were positive for virus by PCR (N = 42). Twenty-eight percent (N = 71) of their offspring were positive for infection, indicating that AgDNV was transmitted between generations, either by vertical transmission or by horizontal transmission from adults to larvae.

Bottom Line: To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes.Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

View Article: PubMed Central - PubMed

Affiliation: The Johns Hopkins Malaria Research Institute, Baltimore, Maryland, United States of America.

ABSTRACT
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs) are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP) in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

Show MeSH
Related in: MedlinePlus