Results

1. Virus isolation from Vero cells
Following inoculation of Vero cells with the nasopharyngeal and oropharyngeal samples, they were observed at 24-hour intervals, and the cytopathic effects were observed from 3 days after inoculation (Figure 1). The inoculated cells were harvested on the 4th day when more than 80% of the cells exhibited cytopathic effects. Virus replication was confirmed using real-time RT-PCR with RNA extracted from the cell culture medium. The Ct values were 14.40 and 18.26 for the nasopharyngeal and oropharyngeal samples, respectively, which were lower than the cycle threshold (Ct) values of 20.85 and 21.85 in the pre-inoculated samples. The number of virus copies in the samples before inoculation was 7.6 × 108 and 3.9 × 108 copy/mL, respectively, and increased by 10–70-fold to 5.4 × 1010 and 4.2 × 109, respectively, in the cell culture supernatants.

2. Analysis of the structure of the virus by electron micrographs
The structure of the virus in the cytoplasm of 3-day post-inoculation cells was examined by electron microscopy (Figure 2). Coronavirus-specific morphology was observed. Virus particle size ranged from 70–90 nm and the virus was observed in a wide range of intracellular organelles, especially in vesicles.

3. Full-length genome and phylogenetic analysis
After inoculating cells with the nasopharyngeal and oropharyngeal samples, RNA was extracted from the virus-replicated cell culture medium. The RNA was amplified with primers for full-length gene analysis, and NGS was performed using Miseq. De novo assembly of the NGS sequence secured 28,818 bp of the full-length gene. The acquired gene was compared with 57 human and animal coronaviruses, including Wuhan/IVDC-HB-01/2019(GISAID accession ID: EPI_ISL_402119~121), which was first reported in Wuhan, 54 reported full-length SARS-CoV-2 genes, Bat-SARS-like CoV, and human SARS-CoV (Figure 3). The analysis showed that the sequence was included in the same cluster as the previously reported SARS-CoV-2 sequence and showed high homology of > 99.5% with other isolated SARS-CoV-2 sequences. The virus was named BetaCoV/Korea/KCDC03/2020, and its full-length gene sequence was registered in WHO GISAID (GISAID accession ID: EPI_ISL_407193).