Fluorescence-based biosensors for West Nile virus detection

Flaviviruses are emerging pathogens, mostly transmitted by arthropod vectors, responsible for human, animal, and zoonotic diseases. The emergence of flaviviruses has been favored in recent decades by factors related to climate change and globalization, which contribute to the arrival and establishment of their vectors in new geographic areas, thus promoting epidemic outbreaks and facilitating these viruses to become endemic in these areas. This is the case of the West Nile virus (WNV) in Europe, which affects its natural bird host populations and also accidental hosts such as humans and horses. Flaviviruses are antigenically related, which induces cross-reactivity, making their serological diagnosis difficult, especially in areas where several flaviviruses co-circulate. Here, we have developed WNV biosensors in which the enzymatic activity of the viral protease, expressed during infection, allows its detection using fluorescence-based techniques. These biosensors carry WNV-specific protease cleavage sites and show high specificity for the detection of both lineages 1 and 2, with limits of detection (LODs) ranging from 0.001-0.0001 MOI at 48 hours post-infection (h.p.i.). These LODs are even lower at 72 h.p.i. reaching as low as an MOI of 2.5 × 10^-6. They are also capable of detecting, although with lower sensitivity, other flaviviruses such as dengue, Zika, and Usutu viruses, without showing reactivity against unrelated viruses. These biosensors have been validated in viral neutralization assays with sera from infected mice and humans, as well as in antiviral screening, with results comparable to those of currently used systems, showing significant potential as clinical and laboratory tools.