Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus

Abstract

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations.

Regulatory RNAs in Bacillus subtilis: A review on regulatory mechanism and applications in synthetic biology

“…To date, toehold switches have primarily been developed and applied in E. coli . There is a likelihood that they could also be adapted for use in B. subtilis synthetic biology and even in diagnostics [ 131 ]. Furthermore, enhancing existing regulatory RNA devices in B. subtilis might involve incorporating novel features inspired by the engineering of other regulatory RNAs.…”

Transcriptome and proteome analysis reveals the anti-cancer properties of Hypnea musciformis marine macroalga extract in liver and intestinal cancer cells

“…1 ug of DNAse-treated RNA was used for first-strand cDNA synthesis (#28,025,013 MMLV reverse transcriptase, Invitrogen) via incubation at 37 °C for 2 h, primed with random hexamers according to manufacturer’s recommendation. RT-qPCR reactions were performed with KAPA SYBRGREEN qPCR mix (Sigma # KR0389_S) as previously described [ 28 ]. All primers used for RT-qPCR are shown in Additional file 1 : Table S1.…”