Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosquitoes.

Three full-length infectious cDNA clones based on the alphavirus chikungunya (CHIKV) were developed and characterized in vitro and in vivo. The full-length clone retained the viral phenotypes of CHIKV in both cell culture and in mosquitoes and should be a valuable tool for the study of virus interactions in an epidemiologically significant natural vector, Aedes aegypti. Two additional infectious clones were constructed that express green fluorescent protein (EGFP) in the midgut, salivary glands, and nervous tissue of Aedes aegypti mosquitoes following oral infection. The two constructs differed in the placement of the subgenomic promoter and the gene encoding EGFP. Viruses derived from the pCHIKic EGFP constructs (5' CHIKV EGFP and 3' CHIKV EGFP) expressed EGFP in 100% of the Ae. aegypti mosquitoes tested on days 7 and 14 post infection (p.i.). The 5' CHIKV EGFP disseminated to 90% of the salivary glands and nervous tissue by day 14 p.i. Dissemination rates of this new viral vector exceeds those of previous systems, thus expanding the repertoire and potential for gene expression studies on this important vector species.