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

Survival rates of sugarfed, bloodfed or SINV-TR339EGFP fed Carb/dcr16 and HWE females. Daily survival rates were monitored for 28 days among one week-old females that had received a non-infectious or SINV-TR339EGFP containing bloodmeal. Sugarfed females were used as control. Bold lines indicate 50% survival.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Survival rates of sugarfed, bloodfed or SINV-TR339EGFP fed Carb/dcr16 and HWE females. Daily survival rates were monitored for 28 days among one week-old females that had received a non-infectious or SINV-TR339EGFP containing bloodmeal. Sugarfed females were used as control. Bold lines indicate 50% survival.

Mentions: We evaluated the effect of midgut-specific impairment of the RNAi pathway on the longevity of mosquitoes once they were infected with SINV-TR339EGFP. So far our data have shown that at 7 days pbm the RNAi pathway-impaired mosquitoes contained higher doses of the virus than the HWE control. We monitored the survival rate of mosquitoes for four weeks after bloodfeeding. Bloodfeeding appeared to have a beneficial effect for both Carb/dcr16 and HWE females since 50% of the insects were still alive at day 25 pbm whereas of the sugarfed control only 20% were alive at the same time point (Fig. 5). When both mosquito strains were infected with SINV-TR339EGFP (titer in the bloodmeal: 2.7 × 107 pfu/ml), their longevity was not affected in comparison to non-infected, bloodfed mosquitoes. The survival curves looked similar for Carb/dcr16 and HWE females, indicating that SINV infection did not cause an obvious fitness cost in the RNAi-impaired mosquitoes.


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)

Survival rates of sugarfed, bloodfed or SINV-TR339EGFP fed Carb/dcr16 and HWE females. Daily survival rates were monitored for 28 days among one week-old females that had received a non-infectious or SINV-TR339EGFP containing bloodmeal. Sugarfed females were used as control. Bold lines indicate 50% survival.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Survival rates of sugarfed, bloodfed or SINV-TR339EGFP fed Carb/dcr16 and HWE females. Daily survival rates were monitored for 28 days among one week-old females that had received a non-infectious or SINV-TR339EGFP containing bloodmeal. Sugarfed females were used as control. Bold lines indicate 50% survival.
Mentions: We evaluated the effect of midgut-specific impairment of the RNAi pathway on the longevity of mosquitoes once they were infected with SINV-TR339EGFP. So far our data have shown that at 7 days pbm the RNAi pathway-impaired mosquitoes contained higher doses of the virus than the HWE control. We monitored the survival rate of mosquitoes for four weeks after bloodfeeding. Bloodfeeding appeared to have a beneficial effect for both Carb/dcr16 and HWE females since 50% of the insects were still alive at day 25 pbm whereas of the sugarfed control only 20% were alive at the same time point (Fig. 5). When both mosquito strains were infected with SINV-TR339EGFP (titer in the bloodmeal: 2.7 × 107 pfu/ml), their longevity was not affected in comparison to non-infected, bloodfed mosquitoes. The survival curves looked similar for Carb/dcr16 and HWE females, indicating that SINV infection did not cause an obvious fitness cost in the RNAi-impaired mosquitoes.

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