Antiviral Hammerhead Ribozyme Strategy for Developing Transgenic Suppression of Arboviruses

Arboviruses, Chikungunya (CHIKV) and Dengue are transmitted by the same major vectors i.e. Aedes aegypti and Aedes albopictus. In this study we first tested a set of seven antiviral hammerhead ribozymes (hRzs) targeting CHIKV structural proteins as potential antivirals. Suppression of CHIKV infectivity was confirmed by challenging hygromycin selected Vero cells transfected with plasmids expressing each of the ribozymes, demonstrating ribozyme #9 and #14 were most effective in inhibiting replication of CHIKV by 2 and 5 orders of magnitude respectively at an MOI of 0.0001. To achieve higher levels of suppression clonals cell populations were generated for each ribozyme enabling complete suppression at 0.0001. Effective clones were selected based on lack of cytopathic effect (CPE). Similar approaches were attempted using maxizyme constructed from hRz#9 and #14. TCID₅₀ analysis confirmed complete suppression of CHIKV replication at an MOI of 0.0001 and 0.001 confirming the advantage of maxizyme over hRzs. The benefits of maxizymes were further confirmed by comparing the suppression potential of clonals expressing Dengue specific maxizymes, connected maxizymes, and hRzs in Vero cells. TCID₅₀ analysis demonstrated complete suppression of Dengue at an MOI of 0.01, compared to only 2 log of suppression with Dengue specific hRzs clonals. All results were further complemented by Caspase-3 assays and quantitative real time PCR assays. This study also showed the usefulness of connected maxizyme over maxizyme, clearly evident based on both TCID₅₀ and quantitative real time PCR analysis. hRzs #9 and #14 exhibiting the best CHIKV suppression were used to generate transgenic mosquitoe line. Both hRzs #9 and #14 promoted by the Aedes aegypti t-RNA^val Pol III promoter were been mobilized into the mosquito genome using the piggybac transposon vector system. Splinkerrtte PCR confirmed the establishment of nine lines with different integration sites in Aedes genome, seven expressing hRz #9 and two expressing hRz #14. Rt-PCR was performed to confirm expression of the hRz transgene in each line.The transgenic mosquitoes expressing either hRz #9 or #14, and control Higgs white eye (HWE) mosquitoes were blood fed over an artificial blood meal containing the attenuated strain of CHIKV 181/25 virus. TCID₅₀ analysis 7 dpi on engorged mosquitoes demonstrated no or minimal viral replication in transgenic mosquitoes compared to control HWE displaying the viral titer of 2x10⁴-10⁵. All of the nine transgenic lines were confirmed as at most partially homozygous using direct PCR analysis. Engineered transgenic mosquitoes having high resistance to CHIKV replication can be potentially utilized in population replacement strategies to achieve elimination of endemic CHIKV.