Seven patients were admitted to a government-approved, nationally designated quarantine ward in Chosun University Hospital between February 2020 and April 2020 and were tested using PCR, viral culture, and immunofluorescence assay (IFA).
The patients were confirmed to be positive using real-time reverse transcription-polymerase chain reaction (RT-PCR) targeting at least two or more genes or who showed > four-fold increase or seroconversion in the antibody titers against SARS-CoV-2.
Sampling And Rna Extraction
Nasopharyngeal and oropharynx swabs and sputum samples were collected using commercial UTM™ kits containing 1 mL of viral transport media (Noble Bio, Korea) and used for RNA extraction. The viral RNA was extracted using a fully automated instrument (PCL South Korea) and Real-prep viral DNA/RNA Kit (Biosewoom, South Korea). RT-PCR was performed to target the NP, E, and RdRp genes, along with a positive reference gene, using distilled water as a negative control. For the NP gene, the primers and probes were designed in-house, whereas for the E and RdRp target genes, Kogene kit (Kogene Biotech Seoul, South Korea) was used; manufacturers’ specifications were used for the reaction conditions and amplifications.
Cell Culture
The African green monkey kidney Vero E6 cell line was purchased from the Korean Cell Line Bank (KCLB no. 21587) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA) and 1X penicillin/streptomycin (PC/SM) at 37°C in a humidified atmosphere of 5% CO2.
Virus Isolation From Clinical Specimens
To isolate SARS-CoV-2 from COVID-19 clinical specimens, samples were prepared by mixing 500 µL of sputum with 200 µL of 100x penicillin/streptomycin (PC/SM) and then incubated at 4°C with a vortex interval of 15 min. After 1 h, the samples were centrifuged at 1,600 × g for 20 min, and the supernatant was collected, to be added to Vero E6 cells.
Vero E6 cells were cultured in 24-well cell culture plates at a density of 2.5 × 105 cells per well for 24 h. The cells were washed with sterile phosphate-buffered saline.
Subsequently, 200 µL of the collected supernatant was added to Vero E6 cells in 500 µL DMEM with 2% FBS and 1X PC/SM and incubated at 37°C for 3–5 days.
The infected Vero E6 cells were checked daily for cytopathic effects (CPE) following the procedures used to detect SARS-CoV and MERS-CoV in previous studies.20,21 CPE was identified when cell rounding, or detachment was observed under a microscope. The cells were scraped from the wells, and 200 µL of cell suspension was transferred to a new 24-well plate with Vero E6 cells in 500 µL medium and incubated at 37°C for 3–5 days (passage 1). According to the protocol described above, the virus isolate underwent two passages. After two passages at intervals of 3–5 days, real-time RT-PCR was performed regardless of the presence or absence of CPE for further quantification of the viral RNA in the scraped cells.
All cell culture infection experiments were performed in a biosafety level-3 laboratory at the Health and Environment Research Institute of Gwangju City.
The whole-genome sequence of SARS-CoV-2 isolates was sequenced using a MiSeq next-generation sequencer with 150PE (Illumina, San Diego, CA, USA), and sequence assembly was performed using Bowtie v.1.1.2 and samtools-mpileup (samtools v.1.9). The whole-genome sequences of the cultured isolates were registered in the Global Initiative for Sharing All Influenza Data (GISAID).
Viral Rna Extraction And Real-time Rt-pcr
Viral RNA extraction and real-time RT-PCR
The virus isolate was confirmed using real-time RT-PCR. Viral RNA was extracted from 200 µL of the cell suspension using a viral DNA/RNA extraction kit (cat no. ZP02201, Taiwan) in an automated nucleic acid purification system (ZiXpress-32) according to the manufacturer’s instructions. RNA was eluted in 100 µL RNase-free water. SARS-CoV-2 was detected using the Bioneer Exicycler 96 Real-time PCR kit (Bioneer Inc., Korea).
Indirect Ifa
To perform the indirect IFA, SARS-CoV-2 samples obtained from the Korea Centers for Disease Control and Prevention were used to infect Vero E6 cells. To prepare the SARS-CoV-2 antigen slide, cells infected for 3 days were cultured on Teflon-coated well slides overnight at 37°C in a 5% CO2 environment and fixed with 80% acetone the next day. The patient serum was diluted using a two-fold serial dilution from 1:16 and then reacted with SARS-CoV-2 antigens in a moist chamber for 30 min at 37°C. After washing, the slides were further incubated with 1:400 diluted secondary antibody (fluorescein isothiocyanate-conjugated anti-human IgM and IgG; MP Biomedicals, OH, USA). The slides were examined using a fluorescence microscope (Olympus IX73, magnification: 400×) after dispensing the mounting solution (Vector Laboratories). An IgG antibody titer of ≥ 1:32 was established as the cut-off using IFA on clinical samples from 15 health-screened individuals.