Superspreading events shaped the Coronavirus Disease 2019 (COVID-19) pandemic, and their rapid identification and containment are essential for disease control. Here we provide a national-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading during the first wave of infections in Austria, a country that played a major role in initial virus transmissions in Europe. Capitalizing on Austria’s well-developed epidemiological surveillance system, we identified major SARS-CoV-2 clusters during the first wave of infections and performed deep whole-genome sequencing of more than 500 virus samples. Phylogenetic-epidemiological analysis enabled the reconstruction of superspreading events and charts a map of tourism-related viral spread originating from Austria in spring 2020. Moreover, we exploited epidemiologically well-defined clusters to quantify SARS-CoV-2 mutational dynamics, including the observation of a low-frequency mutation that progressed to fixation within the infection chain. Time-resolved virus sequencing unveiled viral mutation dynamics within individuals with COVID-19, and epidemiologically validated infector-infectee pairs enabled us to determine an average transmission bottleneck size of 103 SARS-CoV-2 particles. In conclusion, this study illustrates the power of combining epidemiological analysis with deep viral genome sequencing to unravel the spread of SARS-CoV-2, and to gain fundamental insights into mutational dynamics and transmission properties.

Direct type I interferon (IFN) signaling on T cells is necessary for the proper expansion, differentiation, and survival of responding T cells following infection with viruses prominently inducing type I IFN. The reasons for the abortive response of T cells lacking the type I IFN receptor (Ifnar1(-/-)) remain unclear. We report here that Ifnar1(-/-) T cells were highly susceptible to natural killer (NK) cell-mediated killing in a perforin-dependent manner. Depletion of NK cells prior to lymphocytic choriomeningitis virus (LCMV) infection completely restored the early expansion of Ifnar1(-/-) T cells. Ifnar1(-/-) T cells had elevated expression of natural cytotoxicity triggering receptor 1 (NCR1) ligands upon infection, rendering them targets for NCR1 mediated NK cell attack. Thus, direct sensing of type I IFNs by T cells protects them from NK cell killing by regulating the expression of NCR1 ligands, thereby revealing a mechanism by which T cells can evade the potent cytotoxic activity of NK cells.

The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F) 265 is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.Lymphocytic choriomeningitis virus (LCMV) is the prototype of the Arenaviridae family, which includes important human pathogens causing hemorrhagic fever, such as Lassa virus, Junin virus, Machupo virus, and Guanarito virus. LCMV has been widely used as an experimental model for the study of immunology, viral persistence, and viral pathogenesis (34, 50).Virions of LCMV are composed of a nucleocapsid which is surrounded by a lipid envelope containing the envelope glycoprotein (GP). The initial steps in LCMV infection involve the interaction of GP with the cellular receptor of the target cells. Depending on the GP sequence, LCMV uses either alphadystroglycan or an alternative cellular protein as a receptor (15,41,42). After internalization of the virions within vesicles, LCMV GP mediates fusion of the viral and cellular membranes, resulting in delivery of the nucleocapsids into the cytoplasm (9,18,19).LCMV GP is initially expressed as a precursor polypeptide, GP-C, which is posttranslationally cleaved into two subunits, GP-1 and GP-2 (12). Cleavage of LCMV GP-C by a yetunidentified cellular protease occurs in the Golgi or a postGolgi compartment (47), whereas the related Lassa virus GP-C was found to be cleaved early in the secretory pathway by the subtilase SKI-1/S1P (4, 31). The amino-terminal cleavage product GP-1 is a peripheral membrane protein and is noncovalently associated with the carboxy-terminal subunit GP-2, which is an integral membrane protein (14). GP-1 presumably interacts with the cellular receptor, whereas the GP-2 subunit most likely mediates fusion of the viral envelope with the cellular membrane (8,13,26,27). The fusion peptide of GP-2 appears to be activated to a fusion-competent state by a pHdependent conformational change of the LCMV GP subunits (9,18,19).The GP of LCMV and other arenaviruses are cleaved between two uncharged amino acids (13, 30), whereas most other viral GPs are cleaved after a basic amino acid (29). In this study, we analyzed cleavage of LCMV GP in detail. We show that cleavage of LCMV GP is not required for transport to the cell surface but is required for mediating virus infectivity of retroviral pseudotypes. Furthermore, we have determined the consensus motif for cleavage of the LCMV GP, and we show that LCMV GP is cleaved in a late-Golgi or post-Golgi compartment by the cellular subtilase SKI-1/S1P. MATERIALS AND METHODS Construction of LCMV GP and Lassa virus GP expression ...

As the limitations of antiretroviral drug therapy, such as toxicity and resistance, become evident, interest in alternative therapeutic approaches for human immunodeficiency virus (HIV) infection is growing. We developed the first gene therapeutic strategy targeting entry of a broad range of HIV type 1 (HIV-1) variants. Infection was inhibited at the level of membrane fusion by retroviral expression of a membrane-anchored peptide derived from the second heptad repeat of the HIV-1 gp41 transmembrane glycoprotein. To achieve maximal expression and antiviral activity, the peptide itself, the scaffold for presentation of the peptide on the cell surface, and the retroviral vector backbone were optimized. This optimized construct effectively inhibited virus replication in cell lines and primary blood lymphocytes. The membrane-anchored C-peptide was also shown to bind to free gp41 N peptides, suggesting that membrane-anchored antiviral C peptides have a mode of action similar to that of free gp41 C peptides. Preclinical toxicity and efficacy studies of this antiviral vector have been completed, and clinical trials are in preparation.

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseu-