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.

High sequence integrity using CLEVER

A Schematic representation of the alignment of recombinant viruses sequenced by NGS, mutations with a relative abundance of >10% are indicated with a star. A total of 8 bulk (grey) and 5 clonal (green) populations were analyzed.

B Details on substitution and position in the genome. For more detailed analysis see Table S2.

Creating chimeric virus by fragment exchange

A Schematic representation of the exchange of individual fragments. Shown is the replacement of the Wuhan S sequence by the sequence encoding for the Omicron BA.1 or BA.5 S gene. The genetic background (outside of S) is kept in the original Wuhan sequence. All fragments needed to reconstitute the virus were transfected and chimeric virus was rescued.

B Successful rescue of infectious chimeric virus was assessed by CPE formation on Vero E6 cells. Scale bar represents 100 μm.

C Titers of neutralizing antibodies against different SARS-CoV-2 S gene variants were validated in sera from vaccinated patients. Sera were incubated with parental wild-type virus (Wuhan), Omicron BA.1 or BA.5 clinical isolates (BA.1, BA.5) as well as chimeric viruses having the Wuhan background combined with either the Omicron BA.1 S gene (WuhanBA.1 S) or Omicron BA.5 S gene (WuhanBA.5 S). Neutralizing titers were determined with a neutralization assay and TCID50 read-out. Data represents mean ± S.E.M., analyzed with one-way ANOVA followed by Bonferroni’s test (N=5).

Direct mutagenesis using the CLEVER primer design

A Schematic representation of the CLEVER primer design for direct mutagenesis. Shown is the (i) introduction of small nucleotide changes, (ii) the deletion of larger sequences, here shown for ORF3a, and (iii) the insertion of nucleotide stretches such as 3×FLAG as well as a timeline showing the expected work-flow needed from in silico design to virus rescue.

B Details on the G614D and N501Y substitution within the S gene. Shown is position, primer design and the integration into the viral genome confirmed by Sanger sequencing.

C Validation of mutations by Immunoblot. Shown is the validation of the ΔORF3a (left) and ORF8-3×FLAG virus (right). Vero E6 cells were assessed with α-β-actin (α-β-ACT) and viral infection was detected using α-NSP2. ORF3a expression or ORF8/FLAG expression, respectively, were compared to wild-type infected cells and uninfected controls.

D Validation of ΔORF3a by immunocytochemistry. ΔORF3a virus created by direct mutagenesis was compared to its parental wild-type virus. Shown is the expression of ORF3a (magenta) in both viruses. Nucleocapsid (N, green) expression was used to assess viral infection, nuclei were stained with Hoechst (blue).

E Validation of ORF8-3×FLAG by immunocytochemistry. C-terminal tagging of ORF8 with 3×FLAG was achieved with direct mutagenesis. Shown is the expression of ORF8 (magenta) and FLAG (green) in both viruses. Nuclei were stained with Hoechst (blue).

Scalebar is 20µm in D and E.

Direct rescue and mutagenesis of clinical isolates

A Schematic representation of the circular assembly within the eukaryotic cell with the linker fragment. The heterologous elements needed downstream of the 3’ UTR (pA, HDVr, SV40) and upstream of the 5’ UTR (CMV) are assembled in one fragment, separated by a spacer sequence and flanked by homologous regions needed for intracellular recombination.

B Representative agarose gel pictures from PCR fragments amplified by one-step RT-PCR from viral RNA and the linker fragment (L). Recombinant virus was rescued from five (top) or eight fragments (bottom), plus the linker fragment. Asterisks mark fragments harboring the introduced changes within their homology region.

C and D Validation of (C) Omicron BA.5 ΔORF3a and (D) XBB.1.5 ΔORF3a by immunocytochemistry. Shown is the expression of ORF3a (magenta) in Omicron BA.5 and XBB.1.5 clinical isolates and recombinant ΔORF3a viruses. Nucleocapsid (N, green) expression was used to assess viral infection, nuclei were stained with Hoechst (blue). Scale bar represents 20 μm.

Overview of reverse genetics methods for SARS-CoV-2

The most commonly used reverse genetics systems for the rescue of recombinant SARS-CoV-2 are listed and the prominent intermediate steps are depicted. Note that this is a schematic summary and additional steps (such as purification, linearization before transcription, etc.) or small aberrations of the protocol (e.g., different starting material) can apply. The first groups reporting the successful adaptation to SARS-CoV-2 are mentioned. For the CLEVER method, additionally the direct mutagenesis within the initial RT-PCR step is depicted (mutated sites marked with red asterisks). Repeated icons are only labelled once. DNA fragments are represented as blue lines. T7 (T7 RNA polymerase), BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), CPER (circular polymerase extension reaction), ISA (infectious subgenomic amplicons), CLEVER (cloning-free and exchangeable system for virus engineering and rescue).