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Zoonotic viruses pose a global threat to both human and veterinary health, as recently demonstrated by the emergence of SARS-CoV-2, the causative agent of the COVID-19 pandemic. In addition to airborne viruses, highly pathogenic arthropod-borne viruses have emerged globally in recent decades, essentially due to human activities, global warming, habitat destruction, and globalization. An example is the phenuivirus Toscana virus (TOSV). TOSV is a reemerging sandfly-borne enveloped virus causing neuro-invasive infections in humans. Although endemic in the Mediterranean basin, diagnostics, therapeutics, and research on TOSV have been neglected.

Ideally, the prevention of the emergence and spread of emerging pathogens will require approaches that target the first steps of infection in host cells and that block the release of the viral genome into the cytosol, prerequisites for productive infection. This is exactly the topic of this thesis. The PhD work of Jana Koch elucidates the entry mechanisms of two unrelated zoonotic emerging enveloped viruses, SARS-CoV-2 and TOSV, at the cellular and molecular levels. Using fluorescence-based approaches, Jana showed that TOSV traffics along the endosomal machinery in induced pluripotent stem cell-derived human neurons and cell lines, first entering Rab5a+ early endosomes and then Rab7a+ and LAMP1+ late endosomal compartments. TOSV entry required intact late endosomes, from which acid-activated membrane fusion occurred. The pH threshold for fusion was optimal and the fusion was faster at pH 5.5, but fusion also happened with prolonged pre-exposure of viral particles to the slightly acidic pH present in early endosomes. Unexpectedly for a class-II fusion virus, TOSV and other bunyaviruses remained infectious when exposed to low pH in the absence of a target membrane.

In parallel, Jana studied the mechanism of SARS-CoV-2 entry into various cell lines representing the tissues targeted during infection. Jana found that authentic SARS-CoV-2 entered the cytosol from or near the plasma membrane in a rapid, pH-independent manner when host cells expressed the trypsin-like protease TMPRSS2. In contrast, in cells lacking TMPRSS2 expression, SARS-CoV-2 entry was slower and relied on both endosome maturation and acid-dependent endolysosomal cathepsins. Pre-activation of viral particles by proteases bypassed the need for acidification and cathepsin L activity. In addition, I established a microscopy-based cell-cell fusion assay and found that proteolytic processing of S was necessary and sufficient to induce fusion, whereas acidification was not required.

In conclusion, the results of Jana expand our knowledge of the entry of emerging zoonotic viruses. TOSV makes atypical use of endosomal acidity to find its way out of the endocytic machinery, whereas SARS-CoV-2 uses different cellular proteases for membrane fusion and penetration independent of acidification. While the TOSV fusion process itself is triggered by low pH, SARS-CoV-2 requires acidification only for the activity of cathepsins that activate the viral particles. Overall, our study highlights the diversity of strategies developed by viruses to subvert cellular machinery and enter host cells and may provide a basis for the development of antiviral strategies.

Read all about the results of Jana in her articles published in EMBO Journal and PLoS Pathogens