Limits...
The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti.

Khoo CC, Piper J, Sanchez-Vargas I, Olson KE, Franz AW - BMC Microbiol. (2010)

Bottom Line: In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced approximately 50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain.We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level.This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.

ABSTRACT

Background: The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.

Results: As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced approximately 50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes.

Conclusions: We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.

Show MeSH

Related in: MedlinePlus

Transgene design to silence Aa-dcr2 in the midgut of bloodfed females and molecular characterization of transgenic mosquito lines. A) Five hundred base-pair (bp) cDNAs in sense and anti-sense orientations corresponding to a portion of Aa-dcr2 were used for the inverted repeat (IR) construction. Sense and anti-sense cDNA fragments of Aa-dcr2 were separated by the small intron of the Aa-sialonkinin I gene and placed downstream of the Aa-carboxypeptidase A promoter. A transcription termination signal derived from SV40 was added downstream of the IR construct. Numbers below the diagram indicate sizes in bp. Abbreviations: ma. left, ma. right = left, right arms of the Mos1 Mariner transposable element (TE); AeCPA promoter = promoter region of the Ae. aegypti carboxypeptidase A gene; dcr2 = cDNA fragments corresponding to the Aa-dcr2 gene; i = minor intron of the Ae. aegypti sialokinin I gene; svA = transcription termination signal derived from the SV40 virus; EGFP = green fluorescent protein marker; 3xP3 = eye tissue-specific promoter. B) Percentage of midgut-specific silencing of Aa-dcr2 mRNA among nine different transgenic Ae. aegypti lines at 1 day pbm. Aa-dcr2 expression levels in midguts of bloodfed females were normalized for gene expression levels of sugarfed females of the lines at the same time point. Bloodmeals were obtained from mice. Each sample consisted of total RNA from a pool of 20 midguts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2877022&req=5

Figure 1: Transgene design to silence Aa-dcr2 in the midgut of bloodfed females and molecular characterization of transgenic mosquito lines. A) Five hundred base-pair (bp) cDNAs in sense and anti-sense orientations corresponding to a portion of Aa-dcr2 were used for the inverted repeat (IR) construction. Sense and anti-sense cDNA fragments of Aa-dcr2 were separated by the small intron of the Aa-sialonkinin I gene and placed downstream of the Aa-carboxypeptidase A promoter. A transcription termination signal derived from SV40 was added downstream of the IR construct. Numbers below the diagram indicate sizes in bp. Abbreviations: ma. left, ma. right = left, right arms of the Mos1 Mariner transposable element (TE); AeCPA promoter = promoter region of the Ae. aegypti carboxypeptidase A gene; dcr2 = cDNA fragments corresponding to the Aa-dcr2 gene; i = minor intron of the Ae. aegypti sialokinin I gene; svA = transcription termination signal derived from the SV40 virus; EGFP = green fluorescent protein marker; 3xP3 = eye tissue-specific promoter. B) Percentage of midgut-specific silencing of Aa-dcr2 mRNA among nine different transgenic Ae. aegypti lines at 1 day pbm. Aa-dcr2 expression levels in midguts of bloodfed females were normalized for gene expression levels of sugarfed females of the lines at the same time point. Bloodmeals were obtained from mice. Each sample consisted of total RNA from a pool of 20 midguts.

Mentions: We designed a donor plasmid based on the Mariner Mos1 transposable element (TE) containing an Aa-dcr2 IR expression cassette under control of the bloodmeal inducible, midgut-specific AeCPA promoter (Fig. 1A). The donor plasmid was co-injected with a helper plasmid expressing the Mos1 transposase [29] into 1780 pre-blastoderm embryos of the Ae. aegypti HWE strain. The survival rate was 10.3%. After outcrossing to the HWE recipient strain, 115 G0 families were established and their offspring (G1) were screened for eye-specific EGFP expression. We selected 10 different mosquito families that produced transgenic offspring, Carb/dcr16, 29, 44, 54, 69, 79, 113, 125, 126, and 146.


The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti.

Khoo CC, Piper J, Sanchez-Vargas I, Olson KE, Franz AW - BMC Microbiol. (2010)

Transgene design to silence Aa-dcr2 in the midgut of bloodfed females and molecular characterization of transgenic mosquito lines. A) Five hundred base-pair (bp) cDNAs in sense and anti-sense orientations corresponding to a portion of Aa-dcr2 were used for the inverted repeat (IR) construction. Sense and anti-sense cDNA fragments of Aa-dcr2 were separated by the small intron of the Aa-sialonkinin I gene and placed downstream of the Aa-carboxypeptidase A promoter. A transcription termination signal derived from SV40 was added downstream of the IR construct. Numbers below the diagram indicate sizes in bp. Abbreviations: ma. left, ma. right = left, right arms of the Mos1 Mariner transposable element (TE); AeCPA promoter = promoter region of the Ae. aegypti carboxypeptidase A gene; dcr2 = cDNA fragments corresponding to the Aa-dcr2 gene; i = minor intron of the Ae. aegypti sialokinin I gene; svA = transcription termination signal derived from the SV40 virus; EGFP = green fluorescent protein marker; 3xP3 = eye tissue-specific promoter. B) Percentage of midgut-specific silencing of Aa-dcr2 mRNA among nine different transgenic Ae. aegypti lines at 1 day pbm. Aa-dcr2 expression levels in midguts of bloodfed females were normalized for gene expression levels of sugarfed females of the lines at the same time point. Bloodmeals were obtained from mice. Each sample consisted of total RNA from a pool of 20 midguts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2877022&req=5

Figure 1: Transgene design to silence Aa-dcr2 in the midgut of bloodfed females and molecular characterization of transgenic mosquito lines. A) Five hundred base-pair (bp) cDNAs in sense and anti-sense orientations corresponding to a portion of Aa-dcr2 were used for the inverted repeat (IR) construction. Sense and anti-sense cDNA fragments of Aa-dcr2 were separated by the small intron of the Aa-sialonkinin I gene and placed downstream of the Aa-carboxypeptidase A promoter. A transcription termination signal derived from SV40 was added downstream of the IR construct. Numbers below the diagram indicate sizes in bp. Abbreviations: ma. left, ma. right = left, right arms of the Mos1 Mariner transposable element (TE); AeCPA promoter = promoter region of the Ae. aegypti carboxypeptidase A gene; dcr2 = cDNA fragments corresponding to the Aa-dcr2 gene; i = minor intron of the Ae. aegypti sialokinin I gene; svA = transcription termination signal derived from the SV40 virus; EGFP = green fluorescent protein marker; 3xP3 = eye tissue-specific promoter. B) Percentage of midgut-specific silencing of Aa-dcr2 mRNA among nine different transgenic Ae. aegypti lines at 1 day pbm. Aa-dcr2 expression levels in midguts of bloodfed females were normalized for gene expression levels of sugarfed females of the lines at the same time point. Bloodmeals were obtained from mice. Each sample consisted of total RNA from a pool of 20 midguts.
Mentions: We designed a donor plasmid based on the Mariner Mos1 transposable element (TE) containing an Aa-dcr2 IR expression cassette under control of the bloodmeal inducible, midgut-specific AeCPA promoter (Fig. 1A). The donor plasmid was co-injected with a helper plasmid expressing the Mos1 transposase [29] into 1780 pre-blastoderm embryos of the Ae. aegypti HWE strain. The survival rate was 10.3%. After outcrossing to the HWE recipient strain, 115 G0 families were established and their offspring (G1) were screened for eye-specific EGFP expression. We selected 10 different mosquito families that produced transgenic offspring, Carb/dcr16, 29, 44, 54, 69, 79, 113, 125, 126, and 146.

Bottom Line: In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced approximately 50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain.We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level.This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.

ABSTRACT

Background: The RNA interference (RNAi) pathway acts as an innate antiviral immune response in Aedes aegypti, modulating arbovirus infection of mosquitoes. Sindbis virus (SINV; family: Togaviridae, genus: Alphavirus) is an arbovirus that infects Ae. aegypti in the laboratory. SINV strain TR339 encounters a midgut escape barrier (MEB) during infection of Ae. aegypti. The nature of this barrier is not well understood. To investigate the role of the midgut as the central organ determining vector competence for arboviruses, we generated transgenic mosquitoes in which the RNAi pathway was impaired in midgut tissue of bloodfed females. We used these mosquitoes to reveal effects of RNAi impairment in the midgut on SINV replication, midgut infection and dissemination efficiencies, and mosquito longevity.

Results: As a novel tool for studying arbovirus-mosquito interactions, we engineered a transgenic mosquito line with an impaired RNAi pathway in the midgut of bloodfed females by silencing expression of the Aa-dcr2 gene. In midgut tissue of the transgenic Carb/dcr16 line, Aa-dcr2 expression was reduced approximately 50% between 1-7 days post-bloodmeal (pbm) when compared to the recipient mosquito strain. After infection with SINV-TR339EGFP, Aa-dcr2 expression levels were enhanced in both mosquito strains. In the RNAi pathway impaired mosquito strain SINV titers and midgut infection rates were significantly higher at 7 days pbm. There was also a strong tendency for increased virus dissemination rates among the transgenic mosquitoes. Between 7-14 days pbm, SINV was diminished in midgut tissue of the transgenic mosquitoes. Transgenic impairment of the RNAi pathway and/or SINV infection did not affect longevity of the mosquitoes.

Conclusions: We showed that RNAi impaired transgenic mosquitoes are a useful tool for studying arbovirus-mosquito interactions at the molecular level. Following ingestion by Ae. aegypti, the recombinant SINV-TR339EGFP was confronted with both MEB and a midgut infection barrier (MIB). Impairment of the RNAi pathway in the midgut strongly reduced both midgut barriers for the virus. This confirms that the endogenous RNAi pathway of Ae. aegypti modulates vector competence for SINV in the midgut. The RNAi pathway acts as a gatekeeper to the incoming virus by affecting infection rate of the midgut, intensity of infection, and dissemination from the midgut to secondary tissues.

Show MeSH
Related in: MedlinePlus