Journal: Virus Research

Article Title: Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities

doi: 10.1016/j.virusres.2023.199176

Figure Lengend Snippet: Construction and characterization of the Δorf2.4 replicon and Δorf2 replicon. (A) Schema of the two new replicons. The Δorf2 replicon lacks only S (the brown box), while the Δorf2.4 replicon lacks S and E (the green box). (B) Electrophoretic image of the pCC2Fos-Δorf2.4 replicon and pCC2Fos-Δorf2 replicon digested with ZraI. In each of the migrating images, the larger bands are similar to each replicon and the smaller bands to pCC2Fos. The theoretical band sizes of the replicons are 25,927 and 26,179 bp for the Δorf2.4 replicon and Δorf2 replicon, respectively. (C) HiBiT signal kinetics of all three replicons. Huh-7 cells were electroporated with each replicon and the HiBiT signal was measured at the indicated time points. The significance of differences was assessed by a two-way ANOVA, considering p <0.05 as significant (**= p <0.01, ***= p <0.001, ****= p <0.0001). ns means Not Significant. (D) Immunofluorescent assay for viral protein detection. At 48 hpt, NSP8 was stained using the mouse anti-SARS-CoV-2 NSP8 antibody and goat anti-mouse IgG antibody conjugated with Alexa Fluor568 (red-colored cells), and the membrane was stained using the rabbit anti-SARS-CoV-2 membrane antibody and goat anti-rabbit IgG antibody conjugated with the Alexa Fluor 488 antibody (green-colored cells). The nucleus was stained by DAPI. (E) HiBiT luminescence in the supernatant of Δorf2 replicon-transfected cells. Huh-7 cells were electroporated with the Δorf2 replicon and HiBiT luminescence in its supernatant was measured at the indicated timepoints. For panels C and E, the averages and standard errors of at least 2 independent experiments with triplicate samples are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Rabbit anti-SARS-CoV-2 Membrane Glycoprotein polyclonal antibodies (ProSci, Poway, California, USA) and mouse anti-SARS-CoV-2 NSP8 mAb (5A10, GeneTex, Irvine, California, USA) were used in a single mixture for the primary antibody reaction.

Techniques: Staining, Transfection