Figure 1.

Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and spreading by influenza A virus (IAV) defective interfering particles (DIPs). SARS-CoV-2-infected Calu-3 cells (multiplicity of infection (MOI) = 0.03) were treated with IAV DIPs (DI244 or OP7), interferon (IFN)-β, or remdesivir at 1 hour post infection (hpi). For DI244 and OP7 treatment, chromatographically purified and highly concentrated cell culture-derived DIP material [29,30] was used. % (v/v) indicates the fraction with respect to the cell culture volume of 100 µL. Stock concentration, 5.6 × 10⁸ and 1.12 × 10¹¹ defective interfering (DI) viral RNAs (vRNAs)/mL for DI244 and OP7, respectively. (A) Immunofluorescence analysis of the SARS-CoV-2 spike (S) protein expression (green, magenta: DNA) at 3 dpi. Scale bar, 100 µm. (B) Cytopathic effect. Confluence (% of initial) was measured by live-cell microscopy at 2 h intervals. Thick lines represent smoothened data (Savitzky-Golay filter), dotted lines show SD of original data (n = 2, independent experiments). (C) Effective concentration range of DI244 and OP7 compared to IFN-β and remdesivir. Viral titers were determined from the supernatant at 3 days post infection (dpi) by plaque assay. Upper dotted line indicates virus titer in untreated cells, lower dotted line shows the limit of detection (LOD). Independent experiments were conducted; mean ± SD (n = 3) is shown. pfu, plaque-forming units. (D) SARS-CoV-2 growth inhibition by active and inactive DIPs. SARS-CoV-2 infected cells were treated with active or ultraviolet (UV)-irradiated (inactivated) DIPs at 1 hpi. Percentage inhibition of viral growth relative to mock treatment is shown; mean ± SEM (n = 4) is depicted. (E) DIP superinfection 24 h post SARS-CoV-2 infection. Independent experiments were conducted; mean ± SD (n = 2) is shown.