Cell and organoid models to develop new antivirals and diagnostic methods for emerging viruses

Abstract

The ongoing global pandemic of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) is only the latest reminder of the urgent need for new antiviral strategies and diagnostic tools to improve preparedness for emergence of new pathogenic RNA-viruses. A key obstacle for the development of antivirals and diagnostic tools has been the lack of good models to study RNA viruses. This thesis focused on cellular and organoid models for virus infection to facilitate the development of host-targeting antivirals and new diagnostic tools against emerging viruses.

In Paper I, two close analogues from an in-house library of 425 host-targeting compounds were found to have antiviral activity against apathogenic RNA virus Hazara virus (HAZV). These two hit compounds were discovered using a cell- and image-based phenotypic antiviral screening assay. Subsequently, the two compounds were confirmed to have a broad-spectrum antiviral activity against several pathogenic RNA viruses including SARS-CoV-2, Ebola and Crimean-Congo hemorrhagic fever virus. After showing the independence of the compound’s antiviral effect of their originally designed target, human 8-oxoguanine glycosylase 1, thermal protein profiling was used to study the compound target. Applying this technique, the compound was revealed to disturb proteostasis pathways and interactions between cellular heat shock protein 70 complex and viral proteins.

In Paper II, a second antiviral screening of in-house compounds was performed using the previously established image-based phenotypic antiviral screening assay with Zika virus (ZIKV) infected cells. The broad-spectrum activity of the compounds was confirmed by revealing the same compounds to be active against both HAZV and ZIKV. Next, the therapeutic window and antiviral activity of the top-hit compounds was demonstrated in several cellular models of ZIKV infection. Additionally, the novel antiviral compounds showed an antiviral effect and reversed ZIKV induced neurotoxicity in iPS cell derived human brain organoids. Furthermore, mechanism-of-action studies revealed the compound to impair the formation of new virus particles in the late lifecycle steps.

In Paper III, the development of a new diagnostic tool for ZIKV is described using in vitro infected U87 cells and PBMCs. In the method presented, ZIKV cDNA was hybridized using padlock probes and amplified by two rounds of Circle-to-Circle Amplification. Detection was performed using a microfluidic affinity chromatography enrichment platform. Benchmarking of the newly developed method against RT-qPCR, the gold standard diagnostic method for ZIKV detection, confirmed a good correlation between both methods. Altogether, this thesis demonstrates how cell culture tools with varying complexity, unique advantages and challenges can be used to augment the development of novel antiviral drugs and diagnostic methods.