LitCovid-sentences  

PMC:7045880 / 1368-12740 JSONTXT 13 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T17 0-12 Sentence denotes Introduction
T18 13-116 Sentence denotes Coronavirus is an RNA virus consisting of positive-sense single-stranded RNA of approximately 27–32 kb.
T19 117-234 Sentence denotes Coronavirus belong to the family Coronaviridae, which comprises of alpha, beta, delta, and gamma coronaviruses [1,2].
T20 235-382 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 383-477 Sentence denotes The virus is known to infect a wide range of hosts including humans, other mammals, and birds.
T22 478-632 Sentence denotes Infected hosts exhibit different clinical courses, ranging from asymptomatic to severe symptoms in their respiratory, digestive, and genital organs [1,2].
T23 633-706 Sentence denotes There are 6 known coronaviruses that typically cause infection in humans.
T24 707-1012 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 1013-1175 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 1176-1295 Sentence denotes As of February 11th, 2020, a total of 45,000 cases of pneumonia, have been reported from 26 countries, including China.
T27 1296-1429 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 1430-1570 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 1571-1735 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 1736-1796 Sentence denotes The isolated SARS-CoV-2 was named BetaCoV/Korea/KCDC03/2020.
T31 1798-1819 Sentence denotes Materials and Methods
T32 1821-1823 Sentence denotes 1.
T33 1824-1861 Sentence denotes Clinical specimens and RNA extraction
T34 1862-2025 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 2026-2167 Sentence denotes RNA was extracted from clinical samples with a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
T36 2168-2331 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 2333-2335 Sentence denotes 2.
T38 2336-2352 Sentence denotes Real-time RT-PCR
T39 2353-2510 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 2511-2600 Sentence denotes The primer and probe sequences used for RNA-dependent RNA polymerase gene detection were:
T41 2601-2817 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 2818-2969 Sentence denotes 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ (Forward), 5′-ATATTGCAGCAGTACGCACACA-3′ (Reverse) and 5′-ACACTAGCCATCCTTACTGCGCTTCG-3′ (Probe in 5-FAM/3′-BHQ format).
T43 2970-3258 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 3259-3396 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 3397-3550 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 3552-3554 Sentence denotes 3.
T47 3555-3570 Sentence denotes Virus isolation
T48 3571-3672 Sentence denotes The virus was isolated from nasopharyngeal and oropharyngeal samples from putative COVID-19 patients.
T49 3673-3918 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 3919-4087 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 4088-4203 Sentence denotes Virus replication and isolation were confirmed through cytopathic effects, gene detection, and electron microscopy.
T52 4204-4375 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 4377-4379 Sentence denotes 4.
T54 4380-4434 Sentence denotes Next generation sequencing of viral full-length genome
T55 4435-4568 Sentence denotes Using reverse transcriptase, cDNA was synthesized from RNA extracted from the cultured cell medium in which the virus was replicated.
T56 4569-4774 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 4775-4840 Sentence denotes The prepared library was purified and analyzed with Miseq 150 PE.
T58 4841-4942 Sentence denotes De novo assembly was performed on the sequenced product using Megahit to secure a full-length genome.
T59 4944-4946 Sentence denotes 5.
T60 4947-4966 Sentence denotes Sequencing analysis
T61 4967-5028 Sentence denotes Gene sequencing was performed using CLC Main Workbench 7.9.1.
T62 5029-5186 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 5187-5365 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 5367-5369 Sentence denotes 6.
T65 5370-5402 Sentence denotes Transmission electron microscopy
T66 5403-5630 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 5631-5818 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 5819-5920 Sentence denotes After washing 3 times with deionized water, en bloc staining was performed using 0.5% uranyl acetate.
T69 5921-6085 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 6086-6179 Sentence denotes The slides were then embedded in Epon812 plastic resin, and polymerized at 70°C for 48 hours.
T71 6180-6344 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 6345-6489 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 6491-6498 Sentence denotes Results
T74 6500-6502 Sentence denotes 1.
T75 6503-6534 Sentence denotes Virus isolation from Vero cells
T76 6535-6745 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 6746-6858 Sentence denotes The inoculated cells were harvested on the 4th day when more than 80% of the cells exhibited cytopathic effects.
T78 6859-6962 Sentence denotes Virus replication was confirmed using real-time RT-PCR with RNA extracted from the cell culture medium.
T79 6963-7165 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 7166-7382 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 7384-7386 Sentence denotes 2.
T82 7387-7449 Sentence denotes Analysis of the structure of the virus by electron micrographs
T83 7450-7573 Sentence denotes The structure of the virus in the cytoplasm of 3-day post-inoculation cells was examined by electron microscopy (Figure 2).
T84 7574-7619 Sentence denotes Coronavirus-specific morphology was observed.
T85 7620-7756 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 7758-7760 Sentence denotes 3.
T87 7761-7805 Sentence denotes Full-length genome and phylogenetic analysis
T88 7806-7945 Sentence denotes After inoculating cells with the nasopharyngeal and oropharyngeal samples, RNA was extracted from the virus-replicated cell culture medium.
T89 7946-8046 Sentence denotes The RNA was amplified with primers for full-length gene analysis, and NGS was performed using Miseq.
T90 8047-8126 Sentence denotes De novo assembly of the NGS sequence secured 28,818 bp of the full-length gene.
T91 8127-8398 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 8399-8594 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 8595-8743 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 8745-8755 Sentence denotes Discussion
T95 8756-8901 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 8902-9037 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 9038-9148 Sentence denotes The patient had experienced intermittent coughing with sputum 3 days after the administration of antipyretics.
T98 9149-9267 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 9268-9437 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 9438-9669 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 9670-9790 Sentence denotes Further studies are needed to select more sensitive cell lines suitable for virus isolation from low viral load samples.
T102 9791-10070 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 10071-10197 Sentence denotes Results show that the isolate from Korea was clustered with the new coronavirus, SARS-CoV-2 and classified as betacoronavirus.
T104 10198-10376 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 10377-10501 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 10502-10728 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 10729-10892 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 10893-11002 Sentence denotes The Pan-CoV RT-PCR detects all human coronaviruses and animal-derived coronaviruses (personal communication).
T109 11003-11134 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 11135-11220 Sentence denotes Currently, the diagnosis of COVID-19 is based on gene detection via real-time RT-PCR.
T111 11221-11372 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.