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Id Subject Object Predicate Lexical cue
T1 0-76 Sentence denotes Identification of Coronavirus Isolated from a Patient in Korea with COVID-19
T2 78-86 Sentence denotes Abstract
T3 87-97 Sentence denotes Objectives
T4 98-355 Sentence denotes Following reports of patients with unexplained pneumonia at the end of December 2019 in Wuhan, China, the causative agent was identified as coronavirus (SARS-CoV-2), and the 2019 novel coronavirus disease was named COVID-19 by the World Health Organization.
T5 356-497 Sentence denotes Putative patients with COVID-19 have been identified in South Korea, and attempts have been made to isolate the pathogen from these patients.
T6 499-506 Sentence denotes Methods
T7 507-644 Sentence denotes Upper and lower respiratory tract secretion samples from putative patients with COVID-19 were inoculated onto cells to isolate the virus.
T8 645-724 Sentence denotes Full genome sequencing and electron microscopy were used to identify the virus.
T9 726-733 Sentence denotes Results
T10 734-806 Sentence denotes The virus replicated in Vero cells and cytopathic effects were observed.
T11 807-996 Sentence denotes Full genome sequencing showed that the virus genome exhibited sequence homology of more than 99.9% with SARS-CoV-2 which was isolated from patients from other countries, for instance China.
T12 997-1089 Sentence denotes Sequence homology of SARS-CoV-2 with SARS-CoV, and MERS-CoV was 77.5% and 50%, respectively.
T13 1090-1187 Sentence denotes Coronavirus-specific morphology was observed by electron microscopy in virus-infected Vero cells.
T14 1189-1199 Sentence denotes Conclusion
T15 1200-1310 Sentence denotes SARS-CoV-2 was isolated from putative patients with unexplained pneumonia and intermittent coughing and fever.
T16 1311-1366 Sentence denotes The isolated virus was named BetaCoV/Korea/KCDC03/2020.
T17 1368-1380 Sentence denotes Introduction
T18 1381-1484 Sentence denotes Coronavirus is an RNA virus consisting of positive-sense single-stranded RNA of approximately 27–32 kb.
T19 1485-1602 Sentence denotes Coronavirus belong to the family Coronaviridae, which comprises of alpha, beta, delta, and gamma coronaviruses [1,2].
T20 1603-1750 Sentence denotes As the name indicates, the spherical external spike protein displays a characteristic crown shape when observed under an electron microscope [3,4].
T21 1751-1845 Sentence denotes The virus is known to infect a wide range of hosts including humans, other mammals, and birds.
T22 1846-2000 Sentence denotes Infected hosts exhibit different clinical courses, ranging from asymptomatic to severe symptoms in their respiratory, digestive, and genital organs [1,2].
T23 2001-2074 Sentence denotes There are 6 known coronaviruses that typically cause infection in humans.
T24 2075-2380 Sentence denotes Among these, coronavirus 229E, OC43, NL63, and HKU1 generally cause mild cold-like symptoms, whereas severe acute respiratory syndrome-coronavirus (SARS-CoV) in 2003, and Middle East respiratory syndrome-coronavirus (MERS-CoV) in 2012, caused severe respiratory diseases such as pneumonia and death [5,6].
T25 2381-2543 Sentence denotes Following the first outbreaks of unexplained pneumonia in Wuhan, China, in late 2019, a new coronavirus was identified as the causative agent in January 2020 [7].
T26 2544-2663 Sentence denotes As of February 11th, 2020, a total of 45,000 cases of pneumonia, have been reported from 26 countries, including China.
T27 2664-2797 Sentence denotes Approximately 96.8% of all cases have been reported in China, and patients in the Hubei province account for 75% of all cases [8–10].
T28 2798-2938 Sentence denotes In Korea, the first case was reported on January 20, 2020, when SARS-CoV-2 was detected in a traveler entering Korea from Wuhan, China [11].
T29 2939-3103 Sentence denotes This study reports the full genome sequencing of SARS-CoV-2 isolated from putative the 2019 novel coronavirus disease (COVID-19) patients in Korea, by cell culture.
T30 3104-3164 Sentence denotes The isolated SARS-CoV-2 was named BetaCoV/Korea/KCDC03/2020.
T31 3166-3187 Sentence denotes Materials and Methods
T32 3189-3191 Sentence denotes 1.
T33 3192-3229 Sentence denotes Clinical specimens and RNA extraction
T34 3230-3393 Sentence denotes Nasopharyngeal and oropharyngeal swab and sputum samples were collected from symptomatic patients to detect SARS-CoV-2 by real-time reverse transcriptase (RT)-PCR.
T35 3394-3535 Sentence denotes RNA was extracted from clinical samples with a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
T36 3536-3699 Sentence denotes All specimens were handled under a biosafety cabinet according to laboratory biosafety guidelines of Korea Centers for Disease Control and Prevention for COVID-19.
T37 3701-3703 Sentence denotes 2.
T38 3704-3720 Sentence denotes Real-time RT-PCR
T39 3721-3878 Sentence denotes The optimal concentration of primers and probes, which were synthesized using a published sequence [12], was determined with the RNA transcripts of SARS-CoV.
T40 3879-3968 Sentence denotes The primer and probe sequences used for RNA-dependent RNA polymerase gene detection were:
T41 3969-4185 Sentence denotes 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:
T42 4186-4337 Sentence denotes 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ (Forward), 5′-ATATTGCAGCAGTACGCACACA-3′ (Reverse) and 5′-ACACTAGCCATCCTTACTGCGCTTCG-3′ (Probe in 5-FAM/3′-BHQ format).
T43 4338-4626 Sentence denotes 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.
T44 4627-4764 Sentence denotes Reverse transcription was performed at 50°C for 30 minutes, followed by inactivation of the reverse transcriptase at 95°C for 10 minutes.
T45 4765-4918 Sentence denotes 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).
T46 4920-4922 Sentence denotes 3.
T47 4923-4938 Sentence denotes Virus isolation
T48 4939-5040 Sentence denotes The virus was isolated from nasopharyngeal and oropharyngeal samples from putative COVID-19 patients.
T49 5041-5286 Sentence denotes 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.
T50 5287-5455 Sentence denotes 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.
T51 5456-5571 Sentence denotes Virus replication and isolation were confirmed through cytopathic effects, gene detection, and electron microscopy.
T52 5572-5743 Sentence denotes 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.
T53 5745-5747 Sentence denotes 4.
T54 5748-5802 Sentence denotes Next generation sequencing of viral full-length genome
T55 5803-5936 Sentence denotes Using reverse transcriptase, cDNA was synthesized from RNA extracted from the cultured cell medium in which the virus was replicated.
T56 5937-6142 Sentence denotes 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.
T57 6143-6208 Sentence denotes The prepared library was purified and analyzed with Miseq 150 PE.
T58 6209-6310 Sentence denotes De novo assembly was performed on the sequenced product using Megahit to secure a full-length genome.
T59 6312-6314 Sentence denotes 5.
T60 6315-6334 Sentence denotes Sequencing analysis
T61 6335-6396 Sentence denotes Gene sequencing was performed using CLC Main Workbench 7.9.1.
T62 6397-6554 Sentence denotes Alignment was conducted using human and animal coronavirus sequences registered in Global Initiative on Sharing All Influenza Data (GISAID) and NCBI GenBank.
T63 6555-6733 Sentence denotes 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.
T64 6735-6737 Sentence denotes 6.
T65 6738-6770 Sentence denotes Transmission electron microscopy
T66 6771-6998 Sentence denotes 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.
T67 6999-7186 Sentence denotes 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.
T68 7187-7288 Sentence denotes After washing 3 times with deionized water, en bloc staining was performed using 0.5% uranyl acetate.
T69 7289-7453 Sentence denotes Thereafter, 30%, 50%, 70%, 80%, 90%, and 100% ethanol were used sequentially in ascending concentration for dehydration, which was substituted with propylene oxide.
T70 7454-7547 Sentence denotes The slides were then embedded in Epon812 plastic resin, and polymerized at 70°C for 48 hours.
T71 7548-7712 Sentence denotes 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.
T72 7713-7857 Sentence denotes The sections were observed with a transmission electron microscope (Libra120, Carl Zeiss, Germany) at an acceleration voltage of 120 kV [13–15].
T73 7859-7866 Sentence denotes Results
T74 7868-7870 Sentence denotes 1.
T75 7871-7902 Sentence denotes Virus isolation from Vero cells
T76 7903-8113 Sentence denotes 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).
T77 8114-8226 Sentence denotes The inoculated cells were harvested on the 4th day when more than 80% of the cells exhibited cytopathic effects.
T78 8227-8330 Sentence denotes Virus replication was confirmed using real-time RT-PCR with RNA extracted from the cell culture medium.
T79 8331-8533 Sentence denotes 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.
T80 8534-8750 Sentence denotes 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.
T81 8752-8754 Sentence denotes 2.
T82 8755-8817 Sentence denotes Analysis of the structure of the virus by electron micrographs
T83 8818-8941 Sentence denotes The structure of the virus in the cytoplasm of 3-day post-inoculation cells was examined by electron microscopy (Figure 2).
T84 8942-8987 Sentence denotes Coronavirus-specific morphology was observed.
T85 8988-9124 Sentence denotes Virus particle size ranged from 70–90 nm and the virus was observed in a wide range of intracellular organelles, especially in vesicles.
T86 9126-9128 Sentence denotes 3.
T87 9129-9173 Sentence denotes Full-length genome and phylogenetic analysis
T88 9174-9313 Sentence denotes After inoculating cells with the nasopharyngeal and oropharyngeal samples, RNA was extracted from the virus-replicated cell culture medium.
T89 9314-9414 Sentence denotes The RNA was amplified with primers for full-length gene analysis, and NGS was performed using Miseq.
T90 9415-9494 Sentence denotes De novo assembly of the NGS sequence secured 28,818 bp of the full-length gene.
T91 9495-9766 Sentence denotes 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).
T92 9767-9962 Sentence denotes 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.
T93 9963-10111 Sentence denotes 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).
T94 10113-10123 Sentence denotes Discussion
T95 10124-10269 Sentence denotes As of February 12th, 2020, 28 cases of COVID-19 have been reported in Korea, with the first case observed in a traveler residing in Wuhan, China.
T96 10270-10405 Sentence denotes The SARS-CoV-2 was isolated from a Korean patient who had self-administered antipyretics for initial symptoms such as chills and fever.
T97 10406-10516 Sentence denotes The patient had experienced intermittent coughing with sputum 3 days after the administration of antipyretics.
T98 10517-10635 Sentence denotes The SARS-CoV-2 could replicate in other cells (Vero E6 and Caco-II cells), in addition to Vero cells (data not shown).
T99 10636-10805 Sentence denotes The first SARS-CoV-2 was successfully isolated by inoculating human airway epithelial cells with bronchoalveolar-lavage fluid samples from a patient with pneumonia [16].
T100 10806-11037 Sentence denotes Since human airway epithelial cells (because of their resemblance to pseudostratified mucociliary epithelium) require 4–6 weeks to differentiate in vivo, isolation of SARS-CoV-2 using Vero cells or Caco-II cells is more convenient.
T101 11038-11158 Sentence denotes Further studies are needed to select more sensitive cell lines suitable for virus isolation from low viral load samples.
T102 11159-11438 Sentence denotes The sequence of the suspected novel coronavirus (KCDC03) was analyzed using the sequences of 54 SARS-CoV-2 including 6 human coronaviruses, Bat-CoVs, and 51 SARS-CoV-2 that have been registered in GISAID by several countries including China, and a phylogenetic tree was produced.
T103 11439-11565 Sentence denotes Results show that the isolate from Korea was clustered with the new coronavirus, SARS-CoV-2 and classified as betacoronavirus.
T104 11566-11744 Sentence denotes High homology (99.94%–99.99%) was confirmed with the viral sequences reported from other countries, such as those from Wuhan/IVDC-HB-01/2019(GISAID accession ID: EPI_ISL_402119).
T105 11745-11869 Sentence denotes Homology with bat CoV (bat-SL-CoVZC45), SARS-CoV (AY278741) and MERS-CoV (JX869059) was 89.1%, 77.5%, and 50%, respectively.
T106 11870-12096 Sentence denotes However, the lowest homology of 99.5% was observed with BetaCoV/Wuwhan/IVDCHB-04/2020, which could be because of the inaccurate sequence of IVDCHB-04, Hence, it has been excluded from the comparative analysis in other studies.
T107 12097-12260 Sentence denotes Prior to identification of SARS-CoV-2 as the causative agent of the unknown pneumonia in Wuhan, China, pan-CoV RT-PCR was being used to detect SARS-CoV-2 in Korea.
T108 12261-12370 Sentence denotes The Pan-CoV RT-PCR detects all human coronaviruses and animal-derived coronaviruses (personal communication).
T109 12371-12502 Sentence denotes Since the release of the SARS-CoV-2 sequence, a real-time RT-PCR method has been established in the diagnosis of COVID-19 patients.
T110 12503-12588 Sentence denotes Currently, the diagnosis of COVID-19 is based on gene detection via real-time RT-PCR.
T111 12589-12740 Sentence denotes With the isolation of the causative agent, development of serological tests and rapid diagnostic tests in addition to virus detection will be required.
T112 12742-12757 Sentence denotes Acknowledgments
T113 12758-12837 Sentence denotes This study was funded by Korea Centers for Disease Control and Prevention (no.:
T114 12838-12871 Sentence denotes 4800-4837-301 and 4800-4834-303).
T115 12872-12955 Sentence denotes The authors thank Mr. Jun Sub Kim of Korea CDC for his help in sequencing analysis.
T116 12956-12977 Sentence denotes Conflicts of Interest
T117 12978-13031 Sentence denotes The authors have no conflicts of interest to declare.
T118 13033-13148 Sentence denotes Figure 1 Cytopathic effect of SARS-CoV-2 on Vero cells. (A) Mock inoculated cells (B) SARS-CoV-2 inoculated cells.
T119 13149-13232 Sentence denotes Figure 2 Thin section electron micrographs of Vero cells infected with SARS-CoV-2.
T120 13233-13416 Sentence denotes Electron micrographs show representative thin sections of Vero cells infected with SARS-CoV-2; cells were collected at 48 hours after infection for examination by electron microscopy.
T121 13417-13485 Sentence denotes White arrows point to aggregates of assembled intracellular virions.
T122 13486-13611 Sentence denotes Figure 3 Phylogenetic tree analysis of SARS-CoV-2 based on full genome nucleotide sequences using the neighbor-joining tree.
T123 13612-13812 Sentence denotes Values on branches are shown as percentages based on 1,000 bootstrap replicates. (A) Gene analysis of SARS-CoV-2 and other coronaviruses, (B) gene analysis of SARS-CoV-2 and BetaCoV/Korea/KCDC03/2020.