key: cord-1008972-p5ejn9op
authors: Banerjee, Arinjay; Nasir, Jalees A.; Budylowski, Patrick; Yip, Lily; Aftanas, Patryk; Christie, Natasha; Ghalami, Ayoob; Baid, Kaushal; Raphenya, Amogelang R.; Hirota, Jeremy A.; Miller, Matthew S.; Ostrowski, Mario; Kozak, Robert A.; McArthur, Andrew G.; Mossman, Karen; Mubareka, Samira
title: Sequence, infectivity and replication kinetics of SARS-CoV-2 isolated from COVID-19 patients in Canada
date: 2020-04-12
journal: bioRxiv
DOI: 10.1101/2020.04.11.037382
sha: 5bd1016c997085e5cad4bfb58ff9f3ce8d9b62e7
doc_id: 1008972
cord_uid: p5ejn9op

SARS-CoV-2 emerged in December 2019 in Wuhan, China and has since infected over 1.5 million people, of which over 107,000 have died. As SARS-CoV-2 spreads across the planet, speculations remain about the range of human cells that can be infected by SARS-CoV-2. In this study, we report the isolation of SARS-CoV-2 from two COVID-19 patients in Toronto, Canada. We determined the genomic sequences of the two isolates and identified single nucleotide changes in representative populations of our virus stocks. More importantly, we tested a wide range of human immune cells for productive infection with SARS-CoV-2. Here we confirm that human primary peripheral blood mononuclear cells (PBMCs) are not permissive to SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor small nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine cell susceptibility and pathogenicity using in vitro and in vivo infection models.

To detect SARS-CoV-2 in cell culture supernatant, 140 uL of supernatant was removed and 93 detection of viral nucleic acids was performed by RT-PCR using an adaptation of cycling conditions were: 1 cycle of denaturation at 60°C for 10 minutes then 95°C for 2 minutes 105 followed by 44 amplification cycles of 95°C for 10s and 60°C for 15s. Analysis was performed 106 using the Rotor-Gene Q software (Qiagen, https://www.qiagen.com) to determine cycle 107 thresholds (Ct). 108 100µL (400,000 cells) of primary CD4 + , CD8 + , CD19 + and monocytes were washed with 1 ml of 124 phosphate buffered saline (PBS) and spun at 500 g for 5 minutes. The cells were resuspended in 125 100 µL of Live/Dead Violet (ThermoFisher Scientific, https://www.thermofisher.com) as per 126 manufacturer's recommendation and diluted 1:1000 in PBS. Cells were incubated at 4°C for 30 127 minutes. Next, cells were washed with 1 ml of FACS buffer (in-house reagent) and spun at 500 g 128 for 5 minutes. Cells were then stained with 100 µL of their respective stains (aCD4-FITC, 129

aCD8-FITC, aCD19-FITC, aCD14-APC; Biolegend, https://www.biolegend.com) at a 130 concentration of 1µg/mL for 30 min at 4°C. After staining, the cells were washed with 1mL of 131 FACS Buffer and spun at 500 g for 5 minutes. Extra aliquots of cells were left unstained, which 132

were also spun at 500g for 5 minutes. The pellets were resuspended in 100 µL of 1% 133 paraformaldehyde (PFA; ThermoFisher Scientific, https://www.thermofisher.com) and analyzed. 134

Samples were run on the BD LSR Fortessa X20 (BD, https://www.bdbiosciences.com). Cells 135 were gated on Live/Dead negative to exclude debris and dead cells and were then gated on their 136 respective cell surface markers to assess purity. 137 To facilitate virus isolation, we collected mid-turbinate swabs from two COVID-19 patients in 180

Toronto, Canada and transported the swabs in viral transport media to a high containment level 3 181 facility. We inoculated Vero E6 cells with the samples and observed for cytopathic effects (CPE) 182 daily. Seventy-two hours post infection (hpi), cells inoculated with both samples displayed 9 extensive CPE, relative to mock inoculated cells (Figure 1, panel A) . We collected 200 uL of cell 184 culture supernatant and re-infected a fresh layer of Vero E6 cells. Twenty-four hours post 185 infection, both wells containing cells that were re-inoculated displayed extensive CPE (Figure 1,  186 panel B). We extracted viral RNA from the supernatant and confirmed the presence of SARS-187

CoV-2 using a diagnostic quantitative real-time PCR assay (qPCR; Figure 1 , panel C). We also 188 confirmed the presence of coronavirus-like particles in infected Vero E6 cells by electron 189 microscopy ( Figure 1, panel D) . 190 191 Next, we performed genome sequencing of both isolates, generating nearly complete genome 192 sequences with 7500-8000 fold coverage and ~94% completeness, with only ~260 bp and ~200 193 bp at the 5' and 3' termini undetermined (Table 1) In this study, we report the isolation of two replication competent SARS-CoV-2 isolates from 229 COVID-19 patients in Canada. Although we used TPCK-treated trypsin to enhance infection 230 using clinical specimens (Figure 1, panel A) , subsequent infection and virus replication did not 231 require any additional TPCK-treated trypsin (Figure 1, panel B) . We sequenced both isolates to 232 confirm that they were reflective of isolates infecting patients worldwide, selecting SARS-CoV-233 2/ SB3-TYAGNC for experimental investigation as its sequencing produced less minority 234 sequencing reads (Table 1) 

A pneumonia outbreak 276 associated with a new coronavirus of probable bat origin

An interactive web-based dashboard to track COVID-19 in 278 real time. The Lancet Infectious Diseases

Diagnosis and Management of First Case of COVID-19 in Canada: Lessons applied from SARS

Transcriptomic and barrier responses of human airway epithelial cells exposed to cannabis 284 smoke

Positive Selection 286 of a Serine Residue in Bat IRF3 Confers Enhanced Antiviral Protection. iScience

Trimmed Spearman-Karber method for 289 estimating median lethal concentrations in toxicity bioassays

The Method of "Right and Wrong Cases

Gauss's Formula

Interferon Regulatory Factor

Mediated Signaling Limits Middle-East Respiratory Syndrome (MERS) Coronavirus 295

Propagation in Cells from an Insectivorous Bat. Viruses

Detection of 297 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

Multiplex 299 PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly 300 from clinical samples

Cutadapt removes adapter sequences from high-throughput sequencing reads

Trimmomatic: a flexible trimmer for Illumina sequence 304 data

Unicycler: Resolving bacterial genome 309 assemblies from short and long sequencing reads

Fast gapped-read alignment with Bowtie 2

ngsCAT: a tool to 315 assess the efficiency of targeted enrichment sequencing

Identification of mutations in laboratory-evolved microbes 318 from next-generation sequencing data using breseq

SARS-associated coronavirus replication in cell lines. Emerg Infect Dis

Modeling the early events 322 of severe acute respiratory syndrome coronavirus infection in vitro

Respiratory Syndrome Coronavirus 2 from Patient with 2019 Novel Coronavirus Disease, United 326 States. Emerg Infect Dis

Enhanced 328 isolation of SARS-CoV-2 by TMPRSS2-expressing cells

SARS-CoV-2 infects T lymphocytes 331 through its spike protein-mediated membrane fusion

In this study, we show that primary human T cells (CD4 + and CD8 + ) do not support productive 252 virus replication. However, our electron micrographs demonstrate that SARS-CoV-2 likely 253 replicates in CD4 + T cells, but the replication cycle is likely terminated prior to virus maturation 254 and egress. Thus, more work is needed to fully identify the susceptibility and permissivity of 255