Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2

Abstract

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.

Keywords: 2019 novel coronavirus disease; COVID-19; SARS-CoV-2; coronavirus disease; immune cells; isolation; phylogenetics; replication; respiratory infections; severe acute respiratory syndrome coronavirus 2; viruses; zoonoses.

Figure 1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein detection in infected Vero E6 and CD4+ T cells. To detect SARS-CoV-2 protein expression, we infected Vero E6 and CD4+ T cells with SARS-CoV-2 at a multiplicity of infection of 0.1 for 24 h. We immunostained these cells and observed them by using fluorescent microscopy. A) SARS-CoV-2–infected and immunostained Vero E6 cells. B) SARS-CoV-2–infected and immunostained CD4+ T cells. For panels A and B, cells were stained by using an antibody cocktail consisting of SARS-CoV-2 S1 antibody, SARS-CoV-2 N antibody, and diluted serum from a recovered coronavirus disease patient. C) SARS-CoV-2 infected CD4+ T cells immunostained with SARS-CoV-2 S1 antibody (anti-S). Scale bars indicate 400 μm; original magnification ×10.

Figure 2

Isolating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patients with coronavirus disease (COVID-19). A) Vero E6 cells were mock inoculated or inoculated with midturbinate clinical specimens from COVID-19 patients. Cells were incubated for 72 h and observed for cytopathic effect (CPE) under a light microscope. Original magnification ×10. B) To determine if supernatant from Vero E6 cells that were mock inoculated or inoculated with clinical specimens contained replication competent virus, we reinoculated a fresh monolayer of Vero E6 cells and observed cells under a light microscope for CPE after 24 h. Original magnification ×10. C) Quantitative real-time PCR was used to detect SARS-CoV-2 5′-UTR and E gene in RNA extracted from supernatant that was collected from Vero E6 cells that were mock infected or infected with clinical specimens from COVID-19 patients for 72 h. D) Electron micrograph of Vero E6 cells that were reinfected for 48 h with supernatant that was collected from Vero E6 cells infected with clinical specimens. Original magnification ×36,000. Inset, zoomed and cropped from the original electron micrograph, shows coronavirus-like particles. M, mock specimen; specimen 1, SARS-CoV-2/SB2; specimen 2, SARS-CoV-2/SB3-TYAGNC. E, envelope; UTR, untranslated region.

Figure 3

Replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human structural and immune cells. To identify human cells that support SARS-CoV-2 replication, we infected human cell lines and primary cells at a multiplicity of infection of 0.01 (n = 2 independent experiments; supernatant from each experiment was titrated in triplicate). We infected Vero E6 cells as a control. THF (human telomerase life-extended cells) and Calu-3 cells (human lung adenocarcinoma–derived) cells represent human structural cells. THP-1 is a monocyte cell line that was used to derive macrophages and dendritic cells. PBMCs from 2 healthy human donors were used to generate CD4+, CD8+, CD19+, monocytes, and other (CD4–, CD8–, CD19–) cell populations. Supernatant from infected cells was collected at various times and titrated on Vero E6 cells to determine virus titers (TCID₅₀). PBMC, peripheral blood mononuclear cell; TCID₅₀, 50% tissue culture infectious dose.

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 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 indicated by red arrows. A) Vero E6 cells. B) Calu-3 cells. C) CD4+ PBMCs. D) CD8+ PBMCs. E) CD19+ PBMCs. F) Monocytes from PBMCs. G) Other cells from PBMCs (CD4–, CD8–, CD19– cell populations). H) THP-1 monocyte. I) THP-1-derived macrophage. J) THP-1-derived dendritic cell. PBMC, peripheral blood mononuclear cell. Scale bars indicate 200 nm.