Cells and antibodies.
Baby Hamster Kidney 21 cells (BHK-21; ATCC CCL-10) were grown in Minimal Essential Medium (MEM; Life Technologies) with 5% heat-inactivated foetal bovine serum (FBS), 1% L-glutamine (200 Mm; Life Technologies), 5% Tryptose Phosphate Broth (TPB; Life Technologies) and 1% P/S (5000 U/mL; 5 mg/mL). VeroE6 cells (ATCC CRL-1586) were grown in the same medium as BHK-21 cells supplemented with 1% non-essential amino acids (MEM NEAA; Life Technologies). Feline embryonic fibroblast cells (FeA) were grown in Dulbecco's Modified Eagle Medium (DMEM; Life Technologies) with 10% FBS and 1% P/S. FeA cells were kindly provided by VIRBAC laboratory. Feline pulmonary epithelial cells (AK-D; ATCC CCL-150) were grown in a mix of Ham's F12 (F12) and L-15 medium (v/v) with 7% FBS and 1% PS. Additional cell lines and conditions are described in Supplemental Text.
Viral strains.
SARS-CoV-2 was provided courtesy of Pr. Christian Drosten (Charité, Berlin) from the European Archive Collection (human isolate BetaCoV/Germany/BavPat1/2020 p.1; reference: 026V-03883). FeCoV, isolated from a cat died after FIP, was obtained from the American Type Culture Collection (ATCC reference: VR-2126). All experiments were conducted in a BSL3 laboratory.
Virus full genome sequencing.
SARS-CoV-2 and FeCoV clinical samples were first passaged on VeroE6 and FeA respectively. Clarified cell supernatants were used to determine full viral genome sequences. Extraction of nucleic acid was performed using the EZ1 advanced XL machine (Qiagen) with the EZ1 Virus Mini Kit v2.0 (Qiagen). A first random amplification of the viral genome nucleic acids was performed as previously described 14. Amplification was also performed using specific primers and the RT-PCR kit. SuperScript™ IV One-Step RT-PCR System kit (ThermoFisher Scientific) was used. Sequences of primers designed from the full-length sequences are available in Supplemental Table 3. PCR products were purified (Monarch® PCR & DNA Cleanup Kit; New England Biolabs) and pooled in equimolar proportions.
After Qubit quantification using Qubit® dsDNA HS Assay Kit and Qubit 2.0 fluorometer (ThermoFisher Scientific) amplicons were fragmented (sonication) into fragments of 200bp long. Libraries were built adding barcod, for sample identification, and primers to fragmented DNA using AB Library Builder System (ThermoFisher Scientific). To pool equimolarly the barcoded samples a quantification step by real time PCR using Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific) was realised. An emulsion PCR of the pools and loading on 530 chip was realised using the automated Ion Chef instrument (ThermoFisher).
Sequencing was performed using the S5 Ion torrent technology v5.12 (Thermo Fisher Scientific) following manufacturer’s instructions 32. Consensus sequence was obtained after trimming of reads (reads with quality score <0.99, and length <100pb were removed and the 30 first and 30 last nucleotides were removed from the reads) mapping of the reads on a reference (determine following blast of De Novo contigs) using CLC genomics workbench software v.20 (Qiagen). De novo contigs were produced to ensure that the consensus sequence was not affected by the reference sequence and no coronavirus mutated sequences were present in the samples. Quasi species with frequency over 5% were studied.
Preparation of subgenomic cDNA fragments.
For each viruses, eight overlapping fragments were design as showed in. The first and last fragments were directly flanked at their 5’ and 3’extremities by the pCMV and the HDR/SV40pA respectively during de novo synthesis (Genscript manufacturer’s for FeCoV; Thermofisher manufacturer’s for SARS-CoV-2). cDNA were amplified from these de novo synthetic viral fragments as templates. Super Fidelity PCR polymerase kit (Thermofisher Scientific) was used. Primers sequences are described on Supplemental Table 4. The final mixture contained 25 µL of Reaction Mix, 3 µL of DNA (1ng/µL), 100 nM of each primer and 20 µl nuclease-free water. RT-PCR and PCR reactions were performed on a Biometra TProfessional Standard Gradient thermocycler. Amplicons were purified (Monarch® PCR & DNA Cleanup Kit; New England Biolabs) and size of PCR products were verified by gel electrophoresis.
Cell Transfection.
The optimal cell lines for SARS-CoV-2 and FeCoV transfection were determined using different conditions described in Supplemental Table 5. An equimolar mix of subgenomic cDNA fragments of SARS-CoV-2 and FeCoV were transfected into sub-confluent BHK-21 cells and a co-culture of BHK-21 + FeA respectively using Lipofectamine 3000 (ThermoFischer Scientific) for 24 hours. For SARS-CoV-2, a suspension of VeroE6 cells was added 24 hours after transfection and then incubated for 5 days at 37°C, 5% CO2. For FeCoV, fresh medium was added 24 hours after transfection and then incubated for 5 days at 37°C, 5% CO2. Cell supernatant were harvested and serially passaged 2 times to ensure the complete disappearance of the DNA used during the transfection. Passages were performed by inoculating clarified supernatant media onto subconfluent VeroE6 and AK-D cells for SARS- CoV-2 and FeCoV respectively: after 1 h of incubation, cells were washed twice using Hanks' Balanced Salt solution (HBSS; Gibco), fresh medium was added, and plates were incubated 3 days. After the last passage, cell supernatant media were harvested, clarified by centrifugation, aliquoted and stored at -80 ˚C. These virus stocks were used to perform quantification of viral RNA, TCID50 assay, whole-genome sequencing and determined kinetic growth.
Real time RT-PCR assay for virus detection.
Viral RNA was isolated from 100 μL of cell supernatant using a QIAamp Viral RNA kit and RNase-Free DNase Set on the automated QIAcube (Qiagen) automate, following manufacturer’s instructions. Relative quantification of viral RNA was performed using the express One-Step SuperScript® qRT-PCR (Invitrogen). The mixture contained 5 μL of express qPCR SuperMix Universal, 0.25 μL of each primer (500 nM), 0.1 μL of probe (200 nM), 1 μL of express SuperScript® RT mix and 3,5 μL of extracted nucleic acids. Assays were performed using the QuantStudio 12K Flex Real-Time PCR machine (Life technologies) with the following conditions: 50 °C for 15 min, 95°C for 20 sec, followed by 45 cycles of 95°C for 3 s, 60°C for 30 s. Data collection occurred during the 60°C step. The amount of viral RNA was calculated from standard curves using synthetic RNA. Primers used are described in the Supplemental Table 6.
Tissue Culture Infectious Dose 50 (TCID50) assay.
Subconfluent cultures of VeroE6 and AK-D cells in 96-well culture microplate were used for SARS-CoV-2 and FeCoV respectively. Cells were inoculated with 10-fold serial dilutions of clarified cell culture supernatants and incubated 3 days for each viruses. Each row included 6 wells of the dilution and two negative control. The presence of CPE in each well was used to determinate TCID50/mL. The determination of the TCID50/mL for both viruses was performed using the Reed and Muench method 33.
Virus replication kinetics.
Infections at MOI 0.001 and 0.01 were performed using subconfluent VeroE6 or AK-D -cells for SARS-CoV-2 and FeCoV, respectively. Cells were washed twice (HBSS) 4 and 1 hours after the infection with SARS-CoV-2 and FeCoV respectively and fresh medium was added. Cell supernatants were sampled every 12 hours up to 48 hours , respectively, clarified by centrifugation, aliquoted and stored at − 80 °C. They were then analysed using the real-time RT-PCR assay as described above. Each experiment was performed in triplicate.
Statistical analyses.
Exploratory analysis were performed using a two way ANOVA multiple comparisons with Sidak’s multiple comparisons test. Statistical analysis and graphical representation were performed using Graph Pad prism 7.00.