RNA was extracted from the supernatant of Vero E6 cells after 1 passage by using the QIAamp Viral RNA Mini kit (QIAGEN) without addition of carrier RNA. We synthesized double-stranded DNA for sequencing library preparation by using the Liverpool SARS-CoV-2 amplification protocol (11). Two 100-μM primer pools were prepared by combining primer pairs in an alternating fashion to prevent amplification of overlapping regions in a single reaction. In a PCR tube, we added 1 μL Random Primer Mix (ProtoScript II First Strand cDNA Synthesis Kit; New England Biolabs) to 7 μL extracted RNA and denatured it on a SimpliAmp Thermal Cycler (ThermoFisher Scientific) at 65°C for 5 min and then incubated it on ice. We then added 10 μL 2X ProtoScript II Reaction Mix and 2 μL 10X ProtoScript II Enzyme Mix to the denatured sample and performed cDNA synthesis under the following conditions: 25°C for 5 min, 48°C for 15 min, and 80°C for 5 min. After cDNA synthesis, in a new PCR tube we combined 2.5 µL cDNA with 12.5 µL Q5 High-Fidelity 2X Master Mix (New England Biolabs), 8.8 µL nuclease-free water (ThermoFisher Scientific), and 1.125 µL of 100 µM primer pool 1 or 2. PCR cycling was then performed as follows: 98°C for 30 s, followed by 40 cycles of 98°C for 15 s and 65°C for 5 min.
All PCRs were purified by using RNAClean XP (Beckman Coulter,
https://www.beckmancoulter.comExternal Link) at a 1.8× bead-to-amplicon ratio and eluted in 30 μL of RNase-free water (AmericanBio,
https://www.americanbio.comExternal Link). We quantified 2 μL of amplified material by using a Qubit 1X dsDNA assay (ThermoFisher Scientific) according to the manufacturer’s instructions. Illumina sequencing libraries were prepared by using a Nextera DNA Flex Library Prep Kit and Nextera DNA CD Indexes (Illumina,
https://www.illumina.comExternal Link) according to the manufacturer’s instructions. Paired-end 150-bp sequencing was performed for each library on a MiniSeq with a 300-cycle mid-output reagent kit (Illumina), multiplexed with targeted sampling of ≈40,000 clusters per library. Sequencing reads from pools 1 and 2 were combined (as R1 and R2), amplification primer sequences were removed by using Cutadapt version 1.18 (12), and Illumina adaptor sequences were removed and low-quality sequences trimmed or removed by using Trimmomatic (version 0.36) (13). Final sequence quality and confirmation of adaptor/primer trimming were confirmed by using FASTQC version 0.11.5 (14). SARS-CoV-2 genome sequences were assembled by using UniCycler version 0.4.8 (default settings, except for conservative mode) (15) and assembly statistics were generated by QUAST (version 5.0.2) (16). Sequencing depth and completeness of coverage of the assembled genomes was additionally assessed by using Bowtie2 version 2.3.4.1 (17) alignment of the sequencing reads against the assembled contigs, and statistics were generated by ngsCAT (version 0.1) (18). Sequence variation in the assembled genomes was assessed by comparing sequences in BLASTN (
http://blast.ncbi.nlm.nih.gov//Blast.cgiExternal Link) with SARS-CoV-2 genomes available in GenBank as well as BreSeq version 0.35.0 (19) analysis relative to GenBank entry MN908947.3 (first genome sequence reported from the original outbreak in Wuhan). We constructed a phylogenetic tree (Appendix Figure 2) by using maximum-likelihood based on a multiple sequence alignment and RAxML-HPC BlackBox with the general time-reversible plus gamma plus invariate sites model for among-site rate variation (20).
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To determine the replication kinetics of SARS-CoV-2 in human structural and immune cells, we infected Calu-3 cells, THF cells, Vero E6 cells (African green monkey kidney epithelial), THP-1 cells, and primary PBMCs from healthy human donors (CD4+, CD8+, CD19+, monocytes, and other PBMCs; Appendix Figure 1) with an MOI of 0.01. We monitored virus replication in the cell lines for 72 h (Figure 3). We also determined virus replication in PBMCs from healthy donors for 48 h (Figure 3). SARS-CoV-2 propagated to high titers in Vero E6 and Calu-3 cells (Figure 3). SARS-CoV-2 did not replicate efficiently in THF cells (Figure 3). Of note, human immune cell lines and primary PBMCs from healthy donors did not support SARS-CoV-2 replication (Figure 3).
Thumbnail of Electron micrographs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infecting cells. To detect coronavirus-like particles in experimentally infected human structural and immune cells, we infected a range of cells with SARS-CoV-2 at a multiplicity of infection of 0.01 for 48 h. The cells were fixed, processed, and imaged by using a transmission electron microscope (10 fields/cell type). A representative image of each cell type is shown. Virus-like particles are indic
Figure 4. Electron micrographs of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–infected cells. To detect coronavirus-like particles in experimentally infected human structural and immune cells, we infected a range of cells with SARS-CoV-2...
To further support virus replication data, we imaged infected human epithelial, fibroblast, and immune cells by using electron microscopy after 48 h of infection with SARS-CoV-2 at an MOI of 0.01 (Figure 4). We scanned 10 different fields per cell type, each using 4 different magnifications—2,600×, 8,500×, 17,500×, and 36,000×—to determine if the cell populations contained virus-like particles. Virus-like particles were detected in 7/10 fields in Vero E6 cells and 8/10 fields in Calu-3 cells (Figure 4, panels A, B). We also detected virus-like particles in 2/10 fields in primary CD4+ T cells (Figure 4, panel C). We did not observe any virus-like particles in other human immune cells that were experimentally infected with SARS-CoV-2 (Figure 4, panels D–J). To determine if virus-like particles can be detected in Vero E6 cells and PBMCs at earlier time points, we infected these cell populations with SARS-CoV-2 at an MOI of 0.01 and imaged the cells with electron microscopy at 6 h and 12 h after infection (Appendix Figures 3, 4). We observed virus-like particles in 9/10 fields at 6 h after infection and 10/10 fields at 12 h after infection in Vero E6 cells (Appendix Figure 3, panel A, Figure 4, panel A). We also observed virus-like particles in 1/10 fields at 6 h and 1/10 fields at 12 h after infection in CD4+ T cells (Appendix Figure 3, panel B, and Figure 4, panel B). None of the other infected PBMC populations contained detectable virus-like particles (Appendix Figure 3, panels C–F, and Figure 4, panels C–F).
To confirm SARS-CoV-2 infection and protein expression in CD4+ T cells, we infected Vero E6 and CD4+ T cells with SARS-CoV-2 at an MOI of 0.1 for 24 h. We immunostained these cells and observed them by using fluorescent microscopy. To enhance our ability to detect SARS-CoV-2 proteins in these cells, we immunostained the cells by using a cocktail of antibodies that included SARS-CoV-2 S1 antibody, SARS-CoV-2 N antibody, and diluted serum from a recovered COVID-19 patient (Figure 1, panels A and B). We were able to detect SARS-CoV-2 infected Vero E6 and CD4+ T cells by using this antibody cocktail (Figure 1, panels A, B). Furthermore, to confirm SARS-CoV-2 infection of CD4+ T cells by using a single antibody, we immunostained infected CD4+ T cells with anti-SARS-CoV-2 S1 antibody and were able to detect infected cells in the population (Figure 1, panel C).
