Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient

Abstract

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures.

Keywords: 2019 -nCoV; COVID-19; Coronavirus; SARS-CoV-2.

Figure 1.

(A–C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) nasopharyngeal, and (C) oropharyngeal. (D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.

Figure 2.

Viral propagation and quantitation. A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81. B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation. C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.

Figure 3.

Cell lines susceptible to SARS-CoV-2. (A–C) Cell lines were infected with a high MOI of SARS-CoV-2 (MOI >5), washed after adsorption, and subsequently harvested 24 hours post infection for viral titer and protein lysates. A) Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation. B & C) Infected cell protein lysates were probed by western blot with B) rabbit polyclonal anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody. N protein, full length spike (S_FL) and spike S1 (S₁) denoted.

Figure 4.

Multi-step growth curve for SARS-CoV-2. Vero CCL81 (Black) and HUH7.0 cells (green) were infected at MOI 0.1 and cells (solid line) and supernatants (dashed line) were harvested and assayed for viral replication via TCID_50.