PMC:7140597 / 4255-12836 JSON TXT

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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7140597","sourcedb":"PMC","sourceid":"7140597","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7140597","text":"Phylogenetic analysis\nTo analyse the obtained SARS-CoV-2 genomes respectively derived from the infected Chinese tourist (GISAID accession ID: EPI_ISL_412974) and the Italian patient (GISAID accession ID: EPI_ISL_412973) in a phylogenetic context, a dataset of 40 available SARS-Cov-2 complete genomes from different countries was retrieved from GISAID (https://www.gisaid.org/, last access 2 March 2020; Supplementary material). Sequence alignment was performed using MUltiple Sequence Comparison by Log- Expectation (MUSCLE) software (http://www.clustal.org) [6]. Estimation of the best fitting substitution model (Hasegawa, Kishino, and Yano, HKY model) and inference of the phylogenetic tree were conducted by a maximum likelihood approach using Molecular Evolutionary Genetics Analysis across Computing Platforms (MEGA X; https://www.megasoftware.net/) [7]. Support for the tree topology was estimated with 1,000 bootstrap replicates.\nThe maximum likelihood phylogenetic tree in the Figure shows a main clade containing several clusters. The viral genome sequence of the Chinese tourist (GISAID accession ID: EPI_ISL_412974) was identical to that retrieved from one sample of another Chinese tourist, hospitalised at the same hospital in Rome (GISAID accession ID: EPI_ISL_410546). The latter was closely related to that of another sample taken from the same patient (GISAID accession ID: EPI_ISL_410545). These three genome sequences were located in a cluster with genomes mainly from Europe (England, France, Italy, Sweden), but also one from Australia (Figure, highlighted in dark red).\nFigure Phylogenetic analysis of two SARS-CoV-2 complete genome sequences retrieved in this study, with available complete sequences from different countriesa (n = 40 genome sequences)\nGISAID: Global Initiative on Sharing All Influenza Data; HKY: Hasegawa, Kishino, and Yano; MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms; SARS-CoV-2: severe acute respiratory syndrome coronavirus.\nMain clusters are highlighted in different colours. The Wuhan reference genome is in larger font (GenBank accession number: NC_045512.2). The filled circles represent the main supported clusters (bootstrap support values are indicated at the level of the nodes). The scale bar at the bottom of the tree represents 0.000050 nt substitutions per site. The cluster containing the viral sequence of the Chinese tourist who had visited Rome, Italy (GISAID accession ID: EPI_ISL_412974) is in dark red. This cluster includes viral sequences derived from two samples (sputum and nasopharyngeal swabs) of another Chinese tourist visiting Rome (GISAID accession IDs: EPI_ISL_410545 and EPI_ISL_410546). The viral genome sequence (GISAID accession ID: EPI_ISL_412973) derived from a patient from Lombardy, Italy, is in a cluster highlighted in green, which is different from that containing the Chinese tourist’s sequence.\na The tree wasbuilt by using the best fitting substitution model (HKY) through MEGA X software. The genome sequence from the Italian patient in Lombardy (EPI_ISL_412973) appeared in contrast to be located in a different cluster including two genome sequences from Germany (EPI_ISL_406862 Bavaria/Munich and EPI_ISL_412912 Baden-Wuerttemberg-1) and one genome sequence from Mexico (EPI_ISL_ 412972), (Figure, highlighted in green).\nIn the tree, some sequences from other SARS-CoV-2 collected in Europe segregated in separate clusters from the two clusters containing the respective patient sequences characterised in this study. There was for example a cluster formed by two sequences from England and a cluster formed by three sequences from France.\nUsing an alignment, the single nt polymorphisms (SNPs) composition and the potentially resulting variable amino-acids in derived protein sequences compared with the Wuhan reference sequences (MN908947 and NC_045512), were investigated for the genome sequences retrieved in this study, as well as three other genome sequences (EPI_ISL_412972, EPI_ISL_ 412912, EPI_ISL_406862) that clustered with the sequence of the patient in Lombardy.\nThe genome-wide SNPs are reported in Table 1 (positions referred respect to the reference sequence; GenBank accession number: NC_045512). The corresponding amino-acid positions and variations inside the proteins are shown in Table 2.\nTable 1 Single nt polymorphisms (SNPs)a deduced by comparison of two whole genome sequences of SARS-CoV-2 characterised in this studyb with selected SARS-CoV-2 sequences (n = 7 compared sequences)\nSARS-CoV-2 sequence ID (country from which the sequence originated) 241 3037 10265 11083 13206 14408 15806 23403 26144 28881 28882 28883\n5' UTR ORF1ab gene ORF1ab gene ORF 1ab gene ORF1ab gene ORF1ab gene ORF1ab gene Gene S ORF3a gene Gene N Gene N Gene N\nNC_045512 (China) C C G G C C A A G G G G\nMN908947 (China) C C G G C C A A G G G G\nEPI_ISL:412972 (Mexico) T T G G G T - G G A A C\nEPI_ISL: 412912 (Germany) T T A G C T A G G A A C\nEPI_ISL: 406862 (Germany) T T G G C C A G G G G G\nEPI_ISL_412973 (Italy) T T G G C T A G G G G G\nEPI_ISL_412974 (Italy) C C G T C C A A T G G G\nN: nucleocapsid protein; ORF: open reading frame; ORF1ab: ORF encoding polyprotein; S: surface glycoprotein; SARS-CoV-2: severe acute respiratory syndrome coronavirus; SNP: single nt polymorphism; UTR: untranslated region.\na SNPs are shown according to nt positions in the genome sequence and gene location.\nb The two sequences characterised in this study are the ones from Italy (EPI_ISL_412973 and EPI_ISL_412974).\nTable 2 Amino acid variationsa deduced by comparing translations of two whole genome sequences of SARS-CoV-2 characterised in this studyb with those of selected SARS-CoV-2 sequences (n = 7 compared sequences)\nSARS-CoV-2 strains 924 3334 3606 4314 4704 5170 614 251 203 204\nORF1ab ORF1ab ORF1ab ORF1ab ORF1ab ORF1ab Surface glycoprotein ORF3a Nucleocapsid phosphoprotein Nucleocapsid phosphoprotein\nNC_045512 (China) F G L A P Q D G R G\nMN908947 (China) F G L A P Q D G R G\nEPI_ISL:412972 (Mexico) F G L G L -c G G K R\nEPI_ISL: 412912 (Germany) F S L A L Q G G K R\nEPI_ISL: 406862 (Germany) F G L A P Q G G R G\nEPI_ISL_412973 (Italy) F G L A L Q G G R G\nEPI_ISL_412974 (Italy) F G F A P Q D V R G\nORF: open reading frame; ORF1ab: ORF encoding polyprotein; SARS-CoV-2: severe acute respiratory syndrome coronavirus.\na The amino acid positions refer to those in each respective protein sequence of the Wuhan reference (GenBank accession number: MN908947), starting from the first methionine.\nb The two sequences characterised in this study are the ones from Italy (EPI_ISL_412973 and EPI_ISL_412974).\nc -: possible sequencing error. The genome sequence from the Chinese tourist hospitalised in Rome differed in two nt positions from that of the COVID-19 patient in Wuhan (NC_045512), while the genome sequence isolated from the Italian patient showed four nt variations (Table 1).\nFor the sequence of the Chinese tourist, the first SNP inside ORF1ab (bps 3037, AA 924) did not result in an amino acid change.\nIn the Table 2 that depicts five sequences characterised outside of China, overall eight missense mutations can be observed compared to the two reference Wuhan sequences: four locate to the ORF1ab polyprotein, whereby only the mutation L3606F has previously been reported by Phan, 2020 [8]; one, D614G, locates to the surface glycoprotein and has been prior observed [8], but is not in the receptor binding domain (RDB), responsible for virus entry into host cell; one is in the ORF3a protein and two are in the nucleocapsid protein.\nThe sequence of the Chinese tourist hospitalised in Rome on 29 January (EPI_ISL_412974) presented a mutation 3606F in ORF1ab with respect to the reference Wuhan genome (L). In ORF3a, this sequence had a V at amino acid position 251, as opposed to a G in the references from Wuhan.\nMeanwhile, the sequence of the Italian patient from Lombardy (EPI_ISL 412973) presented an L at amino acidic position 4704 with respect to the reference Wuhan genome (P). It also had a mutation in the surface glycoprotein, at amino acidic position 614, where it showed a G compared to the reference sequences from Wuhan that presented a D at that position.\nWith regard to the nucleocapsid protein, both of the sequences from the Italian patient and Chinese tourist presented the same amino acids of the references Wuhan 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PMC:7140597 / 4255-12836 JSONTXT

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PMC:7140597 / 4255-12836 JSON TXT