Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes
ABSTRACT Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.
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ABSTRACT: Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue haemorrhagic fever and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an antipathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti heat shock protein 70 (hsp70) genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ~25-50-fold in whole adults by 4 h after heat-shock, with significant activity (~20-fold) remaining at 24 h. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ~2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of antipathogen molecules.Insect Molecular Biology 12/2011; 21(1):97-106. DOI:10.1111/j.1365-2583.2011.01116.x · 2.98 Impact Factor
Nature 01/1999; 402(6760):370-371. DOI:10.1038/46463 · 42.35 Impact Factor
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ABSTRACT: Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005-2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1-226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.PLoS Pathogens 01/2008; 3(12):e201. DOI:10.1371/journal.ppat.0030201 · 8.06 Impact Factor