PMC:7067204 / 4870-8880 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7067204","sourcedb":"PMC","sourceid":"7067204","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7067204","text":"Materials and methods\n\nViral sequences\nThe complete genome sequence of 2019-nCoV HKU-SZ-005b was available at GenBank (accession no. MN975262) (Table 1). The representative complete genomes of other related βCoVs strains collected from human or mammals were included for comparative analysis. These included strains collected from human, bats, and Himalayan palm civet between 2003 and 2018, with one 229E coronavirus strain as the outgroup.\nTable 1. List of coronaviruses used in this study.\nAccession number Name displayed on the tree Name of full-length genome Year\nAY274119 Human SARS-CoV Tor2 2003 SARS-related coronavirus isolate Tor2 2003\nAY278488 Human SARS-CoV BJ01 2003 SARS coronavirus BJ01 2003\nAY278491 SARS coronavirus HKU-39849 2003 SARS coronavirus HKU-39849 2003 2003\nAY390556 Human SARS-CoV GZ02 2003 SARS coronavirus GZ02 2003\nAY391777 Human CoV OC43 2003 Human coronavirus OC43 2003\nAY515512 Paguma SARS CoV HC/SZ/61/03 2003 SARS coronavirus HC/SZ/61/03 (paguma SARS) 2018\nEF065513 Bat CoV HKU9-1 2006 Bat coronavirus HKU9-1 2006\nFJ588686 Bat SL-CoV Rs672 2006 Bat SARS CoV Rs672/2006 2006\nKC881005 Bat SL-CoV RsSHC014 2013 Bat SARS-like coronavirus RsSHC014 2013\nKC881006 Bat SL-CoV Rs3367 2013 Bat SARS-like coronavirus Rs3367 2013\nKY417146 Bat SL-CoV Rs4231 2016 Bat SARS-like coronavirus isolate Rs4231 2016\nKY417149 Bat SL-CoV Rs4255 2016 Bat SARS-like coronavirus isolate Rs4255 2016\nMG772933 Bat SL-CoV ZC45 2018 Bat SARS-like coronavirus isolate bat-SL-CoVZC45 2018\nMG772934 Bat SL-CoV ZXC21 2018 Bat SARS-like coronavirus isolate bat-SL-CoVZXC21 2018\nMK211377 Bat CoV YN2018C 2018 Coronavirus BtRs-BetaCoV/YN2018C 2018\nMK211378 Bat CoV YN2018D 2018 Coronavirus BtRs-BetaCoV/YN2018Da 2018\nMN975262 HKU-SZ-005b Human 2019-nCoV HKU-SZ-005b 2020\nNC002645 Human CoV 229E 2000 Human coronavirus 229E 2000\nNC006577 Human CoV HKU1 2004 Human coronavirus HKU1 2004\nNC009019 Bat CoV HKU4-1 2006 Bat coronavirus HKU4-1 2006\nNC009020 Bat CoV HKU5-1 2006 Bat coronavirus HKU5-1 2006\nNC014470 Bat SARS-related CoV BM48-31 2009 Bat coronavirus BM48-31/BGR/2008 2008\nNC019843 Human MERS-CoV 2012 Middle East respiratory syndrome coronavirus 2012\naOne nucleotide was added within M gene to maintain the sequence in-frame.\n\nGenome characterization and phylogenetic analysis\nPhylogenetic tree construction by the neighbour joining method was performed using MEGA X software, with bootstrap values being calculated from 1000 trees [12]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [13]. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and were in the units of the number of amino acid substitutions per site [14]. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA X [15]. Multiple alignment was performed using CLUSTAL 2.1 and further visualized using BOXSHADE 3.21. Structural analysis of orf8 was performed using PSI-blast-based secondary structure PREDiction (PSIPRED) [16]. For the prediction of protein secondary structure including beta sheet, alpha helix, and coil, initial amino acid sequences were input and analysed using neural networking and its own algorithm. Predicted structures were visualized and highlighted on the BOXSHADE alignment. Prediction of transmembrane domains was performed using the TMHMM 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/). Secondary structure prediction in the 5′-untranslated region (UTR) and 3′-UTR was performed using the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) with minimum free energy (MFE) and partition function in Fold algorithms and basic options. The human SARS-CoV 5′- and 3′- UTR were used as references to adjust the prediction 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