Rescue and characterization of recombinant SARS-CoV-2
A Schematic representation of the SARS-CoV-2 genome and the ISA-based method for virus recovery. Eight respectively four overlapping fragments covering the whole SARS-CoV-2 genome were PCR amplified. The heterologous CMV promoter was cloned upstream of the 5’ UTR and a poly(A) tail, HDV ribozyme and SV40 termination signal downstream of the 3’ UTR.
B Infectious virus reconstituted from four fragments (rCoV2-4fr) assessed by cytopathic effect (CPE, top) on susceptible Vero E6 cells by supernatant transfer. Plaque size was compared by standard plaque assay two days after inoculation on Vero E6 cells (bottom).
C Workflow for the rescue of recombinant SARS-CoV-2. Four fragments were PCR amplified, mixed in equimolar ratios, transfected into HEK293T cells and infectious virus was recovered 7 days post transfection. Commercially available SARS-CoV-2 antigen quick tests can be used for a rapid non-quantitative analysis.
D Detection of intracellular SARS-CoV-2 nucleocapsid protein (N, green) and nuclei (Hoechst, blue) in Vero E6 cells infected with parental wild-type or recombinant virus by immunocytochemistry.
E Growth kinetics of recombinant virus and its parental wild-type virus. Vero E6 cells were infected in triplicates at an MOI of 0.01, supernatant was collected 12, 24, 48 and 72 hours post infection and analyzed by plaque assay. Cell layers were washed 2 hours post infection. Data represents mean ± S.E.M., analyzed with multiple t-tests and Benjamini, Krieger, and Yekutieli correction (N=3 individual biological replicates, n=3 technical replicates).
F Cryo-transmission electron microscope pictures of parental wild-type virus and recombinant virus in glutaraldehyde-fixed samples.
Scalebar is 100 µm (top) and 2 mm (bottom) in B, 20 µm in D and 100 nm in F.