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. 2023 Oct 24;14:6762. doi: 10.1038/s41467-023-42349-5

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic diagram of IBIS design. IBIS is a live-but-defective SARS-CoV-2 virus. It contains a modified envelope (mE) gene that has three early stop codons (pink lines) introduced to abrogate envelope protein expression. An open-reading frame coding for interferon-beta (IFNβ) was also inserted in place of orf8 (green box). A version of IBIS having mouse IFNβ (mIFNβ) was produced for mouse and hamster experiments. The virus was rescued and propagated in the production cell Vero A9B21, which stably expresses an engineered Envelope transgene and has the STAT1 gene knocked out. b Immunofluorescence staining showing IBIS multiple-round infection in Vero A9B21 but not in parental VeroE6 cells. Parental VeroE6 (left panel) or Vero A9B21 cells (right panel) were either mock-infected or infected with IBIS, SARS-CoV-2 virus harboring mE but without mIFNβ transgene (SARS2-mE), or wildtype SARS-CoV-2 virus (SARS2-wt; ancestral) at MOI 0.1. Twenty‑four hours post-infection, cells were PFA-fixed and immunostained for SARS‑CoV-2 N protein. c Plaque assay of SARS2-mE virus and IBIS in parental VeroE6 and Vero A9B21 cells. SARS2-mE and IBIS viruses were propagated in Vero A9B21, and titered by plaque assay using both parental VeroE6 cells and Vero A9B21 cells. Upper panel shows the titer in plaque-forming unit per milliliter (PFU/mL). Lower panel shows the representative plaque images. d Immunofluorescence staining showing IBIS single-round infection in mIFNβ-responsive cells. Murine fibroblasts L929 stably expressing a human ACE2 (hACE2) transgene were either mock-infected or infected with IBIS, SARS2-mE, or wildtype SARS-CoV-2 virus at MOI 0.1 (left and middle panels). Cells were PFA‑fixed at 24 h post-infection and stained for SARS-CoV-2 N protein. Enlarged images of the selected regions are shown in right panels. Scale bar = 50 µm. The experiments were repeated for 3 times with similar results obtained. Source data are provided as a Source Data file.