Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability. Methylene blue (MB, used for the treatment of methemoglobinemia), Mycophenolic acid (MPA, used in organ transplantation) and the anti-fungal agent Posaconazole (POS) had the broadest anti-CoV spectrum. They inhibited the shedding of SARS-CoV-2 and variants-of-concern (alpha, beta, gamma, delta) from HAEEC in either pre- or post exposure regimens at clinically relevant concentrations. Co-treatment of cultured cells with MB and the FDA-approved SARS-CoV-2 RNA-polymerase inhibitor Remdesivir reduced the effective anti-viral concentrations of MB by 2-fold, and Remdesivir by 4 to 10-fold, indicated by BLISS independence synergy modelling. Neither MB, nor MPA, nor POS affected the cell delivery of SARS-CoV-2 or OC43 (+)sense RNA, but blocked subsequent viral RNA accumulation in cells. Unlike Remdesivir, MB, MPA or POS did not reduce the release of viral RNA in post exposure regimen, thus indicating infection inhibition at a post-replicating step as well. In summary, the data emphasize the power of unbiased, full cycle compound screens to identify and repurpose broadly acting drugs against coronaviruses.

Imaging across scales reveals disease mechanisms in organisms, tissues, and cells. Yet, particular infection phenotypes, such as virus-induced cell lysis, have remained difficult to study. Here, we developed imaging modalities and deep learning procedures to identify herpesvirus and adenovirus (AdV) infected cells without virusspecific stainings. Fluorescence microscopy of vital DNA-dyes and live-cell imaging revealed learnable virus-specific nuclear patterns transferable to related viruses of the same family. Deep learning predicted two major AdV infection outcomes, nonlytic (nonspreading) and lytic (spreading) infections, up to about 20 hr prior to cell lysis. Using these predictive algorithms, lytic and non-lytic nuclei had the same levels of green fluorescent protein (GFP)-tagged virion proteins but lytic nuclei enriched the virion proteins faster, and collapsed more extensively upon laser-rupture than non-lytic nuclei, revealing impaired mechanical properties of lytic nuclei. Our algorithms may be used to infer infection phenotypes of emerging viruses, enhance single cell biology, and facilitate differential diagnosis of non-lytic and lytic infections.

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Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance, and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endo-lysosomes. Serial passaging of RV-A16 in presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endo-lysosomotropic agent ammonium chloride (NH 4 Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in the viral proteins 1 and 3 (VP1, VP3), A2526G (serine 66 to asparagine; S66N), and G2274U (cysteine 220 to phenylalanine; C220F), respectively. Both mutations conferred cross-resistance to BafA1, NH 4 Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature and soluble intercellular adhesion molecule-1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. Importance Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs or vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants, and an immense diversity of antigens in more than 160 different RV types. Here, we provide insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endo-lysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

artificial intelligence | machine learning | deep learning | predictive infection | computational image analyses | single cell analyses | nucleus | live cell DNA stain | nuclear rupture | nuclear pressure | nuclear envelope | live cell imaging | fluorescence microscopy | laser ablation | adenovirus | herpes simplex virus | virus spreading | virus egress | oncolytic virus | cell lysis | lytic infectionCorrespondence: urs.greber@imls.uzh.ch