Severe Acute Respiratory Syndrome Coronavirus 2 From Patient With Coronavirus Disease, United States

Jennifer Harcourt; Azaibi Tamin; Xiaoyan Lu; Shifaq Kamili; Senthil K. Sakthivel; Janna Murray; Krista Queen; Ying Tao; Clinton R. Paden; Jing Zhang; Yan Li; Anna Uehara; Haibin Wang; Cynthia Goldsmith; Hannah A. Bullock; Lijuan Wang; Brett Whitaker; Brian Lynch; Rashi Gautam; Craig Schindewolf; Kumari G. Lokugamage; Dionna Scharton; Jessica A. Plante; Divya Mirchandani; Steven G. Widen; Krishna Narayanan; Shinji Makino; Thomas G. Ksiazek; Kenneth S. Plante; Scott C. Weaver; Stephen Lindstrom; Suxiang Tong; Vineet D. Menachery; Natalie J. Thornburg

Disclosures

Emerging Infectious Diseases. 2020;26(6):1266-1273. 

In This Article

Methods

Specimen Collection

Virus isolation from patient samples was deemed not to be human subjects research by the National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention (CDC) (research determination no. 0900f3eb81ab4b6e). Clinical specimens from a case-patient who had acquired COVID-19 during travel to China and who was identified in Washington, USA, were collected as described.[1] Nasopharyngeal (NP) and oropharyngeal (OP) swab specimens were collected on day 3 postsymptom onset, placed in 2–3 mL of viral transport medium, used for molecular diagnosis, and frozen. Confirmed PCR-positive specimens were aliquoted and refrozen until virus isolation was initiated.

Cell Culture, Limiting Dilution, and Virus Isolation

We used Vero CCL-81 cells for isolation and initial passage. We cultured Vero E6, Vero CCL-81, HUH 7.0, 293T, A549, and EFKB3 cells in Dulbecco minimal essential medium (DMEM) supplemented with heat-inactivated fetal bovine serum (5% or 10%) and antibiotics/antimycotics (GIBCO, https://www.thermofisher.com). We used both NP and OP swab specimens for virus isolation. For isolation, limiting dilution, and passage 1 of the virus, we pipetted 50 μL of serum-free DMEM into columns 2–12 of a 96-well tissue culture plate, then pipetted 100 μL of clinical specimens into column 1 and serially diluted 2-fold across the plate. We then trypsinized and resuspended Vero cells in DMEM containing 10% fetal bovine serum, 2× penicillin/streptomycin, 2× antibiotics/antimycotics, and 2× amphotericin B at a concentration of 2.5 × 105 cells/mL. We added 100 μL of cell suspension directly to the clinical specimen dilutions and mixed gently by pipetting. We then grew the inoculated cultures in a humidified 37°C incubator in an atmosphere of 5% CO2 and observed for cytopathic effects (CPEs) daily. We used standard plaque assays for SARS-CoV-2, which were based on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) protocols.[9,10]

When CPEs were observed, we scraped cell monolayers with the back of a pipette tip. We used 50 μL of viral lysate for total nucleic acid extraction for confirmatory testing and sequencing. We also used 50 μL of virus lysate to inoculate a well of a 90% confluent 24-well plate.

Inclusivity/Exclusivity Testing

From the wells in which CPEs were observed, we performed confirmatory testing by using real-time reverse transcription PCR (CDC) and full-genome sequencing.[1] The CDC molecular diagnostic assay targets 3 portions of the nucleocapsid gene, and results for all 3 portions must be positive for a sample to be considered positive (https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html and https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). To confirm that no other respiratory viruses were present, we performed Fast Track Respiratory Pathogens 33 Testing (FTD Diagnostics, http://www.fast-trackdiagnostics.com).

Whole-genome Sequencing

We designed 37 pairs of nested PCRs spanning the genome on the basis of the coronavirus reference sequence (GenBank accession no. NC045512). We extracted nucleic acid from isolates and amplified by using the 37 individual nested PCRs. We used positive PCR amplicons individually for subsequent Sanger sequencing and also pooled them for library preparation by using a ligation sequencing kit (Oxford Nanopore Technologies, https://nanoporetech.com), subsequently for Oxford Nanopore MinION sequencing. We generated consensus nanopore sequences by using Minimap version 2.17 (https://github.com) and Samtools version 1.9 (http://www.htslib.org). We generated consensus sequences by Sanger sequencing from both directions by using Sequencher version 5.4.6 (https://www.genecodes.com), and further confirmed them by using consensus sequences generated from nanopore sequencing.

To sequence passage 4 stock, we prepared libraries for sequencing by using the Next Ultra II RNA Prep Kit (New England Biolabs, https://www.neb.com) according to the manufacturer's protocol. In brief, we fragmented ≈70–100 ng of RNA for 15 min, followed by cDNA synthesis, end repair, and adaptor ligation. After 6 rounds of PCR, we analyzed libraries by using an Agilent Bioanalyzer (https://www.agilent.com) and quantified them by using a quantitative PCR. We pooled samples and sequenced samples by using a paired-end 75-base protocol on an Illumina (Illumina, Inc., https://www.illumina.com) MiniSeq instrument and using the High-Output Kit and then processed reads by using Trimmomatic version 0.36[11] to remove low-quality base calls and any adaptor sequences. We used the de novo assembly program ABySS[12] to assemble the reads into contigs by using several different sets of reads and kmer values ranging from 20 to 40. We compared contigs >400 bases against the National Center for Biotechnology Information (Bethesda, MD, USA) nucleotide collection using BLAST (https://blast.ncbi.nlm.nih.gov). A nearly full-length viral contig obtained in each sample had 100% identity to the 2019-nCoV/USA-WA1/2020 strain (GenBank accession no. MN985325.1). All the remaining contigs mapped to either host cell rRNA or mitochondria. We mapped the trimmed reads to the reference sequence by using BWA version 0.7.17[13] and visualized these reads by using the Integrated Genomics Viewer[14] to confirm the identity with the USA-WA1/2020 strain.

Electron Microscopy

We scraped infected Vero cells from the flask, pelleted by low-speed centrifugation, rinsed with 0.1 mol/L phosphate buffer, pelleted again, and fixed for 2 h in 2.5% buffered glutaraldehyde. We then postfixed specimens with 1% osmium tetroxide, en bloc stained with 4% uranyl acetate, dehydrated, and embedded in epoxy resin. We cut ultrathin sections, stained them with 4% uranyl acetate and lead citrate, and examined them by using a Thermo Fisher/FEI Tecnai Spirit electron microscope (https://www.fei.com).

Protein Analysis and Western Blotting

We harvested cell lysates by using Laemmli sodium dodecyl sulfate–polyacrylamide gel electrophoresis sample buffer (Bio-Rad, https://www.bio-rad.com) containing 2% SDS and 5% β-mercaptoethanol. We removed the cell lysates from a Biosafety Level 3 Laboratory, boiled them, and load them onto a polyacrylamide gel. We subjected the lysates to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride polyvinylidene fluoride membrane. We then blocked the membrane in 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h, followed by a short wash with TBS-T. We incubated the membrane overnight with primary antibody, either rabbit polyclonal serum against the SARS-CoV spike protein (#40150-T52; Sino Biological, https://www.sinobiological.com), β-actin antibody (#4970; Cell Signaling Technology, https://www.cellsignal.com), or a custom rabbit polyclonal serum against SARS-CoV nucleocapsid. We then washed the membrane with 3 times with TBS-T and applied horseradish peroxidase-conjugated secondary antibody for 1 h. Subsequently, we washed the membrane 3 times with TBS-T, incubated with Clarity Western ECL Substrate (#1705060S; Bio-Rad), and imaged with a multipurpose imaging system.

Generation of SARS-CoV Nucleocapsid Antibodies

We used the plasmid pBM302[15] to express SARS-CoV nucleocapsid protein, with a C-terminal His6 tag, to high levels within the inclusion bodies of Escherichia coli and the recombinant protein was purified from the inclusion bodies by using nickel-affinity column chromatography under denaturing conditions. We used stepwise dialysis against Tris/phosphate buffer to refold the recombinant SARS-CoV nucleocapsid protein with decreasing concentrations of urea to renature the protein. We then immunized rabbits with the renatured, full-length, SARS-CoV nucleocapsid protein to generate an affinity-purified rabbit anti–SARS-CoV nucleocapsid protein polyclonal antibody.

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