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Novel fluorescent cell-based sensors for detection of viral pathogens
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Diseases caused by viruses are responsible for millions of deaths worldwide. However, viruses are used for development of virus-based biopharmaceuticals (VBBs), like vaccines and viral vectors to treat/prevent diseases. These applications require reliable methods for virus and viral vectors quantification. Nevertheless, current titration techniques fail to provide fast, reliable method with high-throughput.
This thesis aimed at developing genetically encoded fluorescent cell-based sensors for virus detection - VISENSORS. These are activated upon recognition by viral proteases (VP) of specific cleavable sequences, allowing detection of label-free virus and viral vectors. Three strategies were developed based on split-Green Fluorescent Protein (GFP) fluorescence inhibition caused by inducing a structural distortion (SD): coiled-coil, embedded and cyclized split-GFP VISENSORS. After VP proteolysis, SD is relieved, and fluorescence is restored.
Different backbones were optimized per strategy, and their performance evaluated under VP activity for detection of Adenovirus and Human Immunodeficiency Virus type one (HIV-1) by transient screenings and latter, stably expressing VISENSORS in mammalian cells and analysing its response to viral infection.
The coiled coil strategy for Adenovirus-VISENSOR showed the lowest performance being only tested in transient. The cyclized and embedded strategies were tested upon adenovirus infection, the first exhibited a lower Signal/Noise ratio (S/N) (1.6) possibly caused by sensor instability, the latter showed promising performance - S/N of 2.0 - with room for enhancement through improving SD.
HIV-1-VISENSORS were successfully established, where embedded and cyclized strategies proved similar S/N performances in transient.
This work contributes for the optimization of Adenovirus and HIV-1 label-free sensors, by analysing the impact of SD strategies and VP cleavable sequences, showing these have high impact in sensor performance. Embedded strategy showed potential although further improvements to reduce the Noise are needed
VISENSORS can be adapted to different viruses for detection and quantification of viruses and VBBs.
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Biossensores fluorescentes deteção e quantificação de vírus sensores baseados em células adenovírus vírus humano da imunodeficiência tipo um