Materials and Methods

1. Clinical specimens and RNA extraction
Nasopharyngeal and oropharyngeal swab and sputum samples were collected from symptomatic patients to detect SARS-CoV-2 by real-time reverse transcriptase (RT)-PCR. RNA was extracted from clinical samples with a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. All specimens were handled under a biosafety cabinet according to laboratory biosafety guidelines of Korea Centers for Disease Control and Prevention for COVID-19.

2. Real-time RT-PCR
The optimal concentration of primers and probes, which were synthesized using a published sequence [12], was determined with the RNA transcripts of SARS-CoV. The primer and probe sequences used for RNA-dependent RNA polymerase gene detection were: 5′-GTGARATGGTCATGTGTGGCGG-3′ (Forward), 5′-CARATGTTAAASACACTATTAGCATA-3′ (Reverse) and 5′-CAGGTGGAACCTCATCAGGAGATGC-3′ (Probe in 5-FAM/3′-BHQ format) and the primer and probe sequences used for E gene detection were: 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ (Forward), 5′-ATATTGCAGCAGTACGCACACA-3′ (Reverse) and 5′-ACACTAGCCATCCTTACTGCGCTTCG-3′ (Probe in 5-FAM/3′-BHQ format). A 25-μL reaction was setup that contained 5 μL of RNA, 12.5 μL of 2 × reaction buffer provided with the Agpath IDTM 1 step RT-PCR system (Thermo Fisher Scientific, Waltham, USA), 1 μL of 25 × enzyme mixture, 1 μL of forward and reverse primers at 10 pM, and 0.5 μL of each probe at 10 pM. Reverse transcription was performed at 50°C for 30 minutes, followed by inactivation of the reverse transcriptase at 95°C for 10 minutes. PCR amplification was performed with 40 cycles at 95°C for 15 seconds and 60°C for 1 minute using an ABI 7500 Fast instrument (Thermo Fisher Scientific).

3. Virus isolation
The virus was isolated from nasopharyngeal and oropharyngeal samples from putative COVID-19 patients. Oropharyngeal samples were diluted with viral transfer medium containing nasopharyngeal swabs and antibiotics (Nystadin, penicillin-streptomycin 1:1 dilution) at 1:4 ratio and incubated for 1 hour at 4°C, before being inoculated onto Vero cells. Inoculated Vero cells were cultured at 37°C, 5% CO2 in 1× Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% fetal bovine serum and penicillin-streptomycin. Virus replication and isolation were confirmed through cytopathic effects, gene detection, and electron microscopy. Viral culture of SARS-CoV-2 was conducted in a biosafety Level-3 facility according to laboratory biosafety guidelines of Korea Centers for Disease Control and Prevention.

4. Next generation sequencing of viral full-length genome
Using reverse transcriptase, cDNA was synthesized from RNA extracted from the cultured cell medium in which the virus was replicated. A next generation sequencing (NGS) library was constructed after amplifying the full-length genes of the isolates using the synthesized cDNA and primers designed based on published SARS-CoV-2 DNA sequence. The prepared library was purified and analyzed with Miseq 150 PE. De novo assembly was performed on the sequenced product using Megahit to secure a full-length genome.

5. Sequencing analysis
Gene sequencing was performed using CLC Main Workbench 7.9.1. Alignment was conducted using human and animal coronavirus sequences registered in Global Initiative on Sharing All Influenza Data (GISAID) and NCBI GenBank. The phylogenetic tree was analyzed using MEGA6 with the neighbor-joining method, maximum composite likelihood-parameter distance matrix, and bootstrap values of 1,000 replicates.

6. Transmission electron microscopy
For transmission electron microscopy, the inoculated cells were prefixed by incubating in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) to prevent the autolysis of the cells infected with virus. To minimize the chemical reaction between pre- and post-fixation, the slides were washed 3 times using the same buffer as in the fixative solution and post-fixed with 1% osmium tetroxide. After washing 3 times with deionized water, en bloc staining was performed using 0.5% uranyl acetate. Thereafter, 30%, 50%, 70%, 80%, 90%, and 100% ethanol were used sequentially in ascending concentration for dehydration, which was substituted with propylene oxide. The slides were then embedded in Epon812 plastic resin, and polymerized at 70°C for 48 hours. The prepared plastic block was cut to 70-nm thick sections using an ultramicrotome and mounted on a 100-mesh nickel grid, and electrostained with 5% uranyl acetate. The sections were observed with a transmission electron microscope (Libra120, Carl Zeiss, Germany) at an acceleration voltage of 120 kV [13–15].