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PMC:7140597 JSONTXT 24 Projects

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Id Subject Object Predicate Lexical cue
T1 0-189 Sentence denotes Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe
T2 191-199 Sentence denotes Abstract
T3 200-368 Sentence denotes Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere.
T4 369-531 Sentence denotes In a phylogenetic tree, the Italian patient’s sequence clustered with sequences from Germany while the tourist’s sequence clustered with other European sequences.
T5 532-650 Sentence denotes Some additional European sequences in the tree segregated outside the two clusters containing the patients’ sequences.
T6 651-747 Sentence denotes This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.
T7 749-1047 Sentence denotes An outbreak of a viral respiratory illness (officially named by the World Health Organization coronavirus disease, COVID-19) caused by the newly discovered severe acute respiratory syndrome coronavirus (SARS-CoV-2), started around mid-December 2019, in the city of Wuhan, Hubei province, China [1].
T8 1048-1206 Sentence denotes The outbreak subsequently spread further and as at 31 March 2020, 750,890 cases of COVID-19 have been confirmed worldwide including 668,345 outside China [2].
T9 1207-1407 Sentence denotes Since 20 February 2020, sustained local transmission has been documented in Italy [3], where to date, 98,716 COVID-19 cases testing positive for SARS-CoV-2 have been diagnosed, with 10,943 deaths [4].
T10 1408-1627 Sentence denotes To gain further understanding on the molecular epidemiology of the outbreak in Italy, we characterised the full-genome sequence of two SARS-CoV-2 strains respectively isolated from two patients diagnosed in the country.
T11 1628-1839 Sentence denotes The first patient was a Chinese tourist from Wuhan diagnosed at the end of January, who had visited Rome and not been in areas of Italy later found to be the initially affected areas of the epidemic in Lombardy.
T12 1840-2003 Sentence denotes The second patient was an Italian person, with no apparent direct epidemiological link with China and who was diagnosed in the second half of February in Lombardy.
T13 2004-2118 Sentence denotes The sequences presented are analysed in the context of other available genome sequences from Europe and elsewhere.
T14 2120-2175 Sentence denotes Patients, virus cultivation and whole genome sequencing
T15 2176-2370 Sentence denotes The two patients in this study had both been hospitalised with an acute respiratory illness (pneumonia), showing a bilateral lung involvement with ground-glass opacity, requiring intensive care.
T16 2371-2481 Sentence denotes The Chinese patient had had onset of symptoms on 29 January 2020 and had been diagnosed in a hospital in Rome.
T17 2482-2601 Sentence denotes The Italian patient whose onset of symptoms had occurred on 10 February 2020 had been diagnosed in a hospital in Milan.
T18 2602-2781 Sentence denotes Biological samples from both patients had been confirmed as being SARS-CoV-2 positive by the National Reference Laboratory (NRL) of the Istituto Superiore di Sanità (ISS) in Rome.
T19 2782-2840 Sentence denotes The samples used for this study were nasopharyngeal swabs.
T20 2841-3017 Sentence denotes These had been respectively sampled on the same day of hospitalisation, when symptoms occurred, for the Chinese tourist and 10 days after symptom onset for the Italian patient.
T21 3018-3225 Sentence denotes An aliquot of each patient’s nasopharyngeal sample was used to generate in vitro cultures in Vero cells grown in modified Eagle’s medium (MEM; Gibco, Thermofisher, United Kingdom) supplemented with GlutaMAX.
T22 3226-3369 Sentence denotes A total of 140 µL of each culture’s supernatant was used for viral RNA extraction using the QIAMP VIRAL RNA mini kit (Qiagen, Hilden, Germany).
T23 3370-3656 Sentence denotes The obtained genomic RNAs were retro-transcribed using the SuperScript III Reverse Transcriptase kit (Invitrogen, Carisbad, United States (US)) and double-stranded DNAs were subsequently obtained by Klenow enzyme (Roche, Basel, Switzerland) according to the manufacturer’s instructions.
T24 3657-3874 Sentence denotes The Nextera XT kit was used for library preparations and whole genome sequencing was performed using the Illumina Miseq Reagent Nano Kit, V2 (2 x 150 cycles) on the Illumina MiSeq instrument (Illumina, San Diego, US).
T25 3875-4072 Sentence denotes The reads were trimmed for quality and length and assembled by mapping to the reference genome from Wuhan, China (GenBank accession number: NC_045512.2) using Geneious Prime (www.geneious.com) [5].
T26 4073-4253 Sentence denotes Viral sequences from the two patients were deposited in the Global Initiative on Sharing All Influenza Data (GISAID; https://www.gisaid.org/epiflu-applications/next-hcov-19-app/ ).
T27 4255-4276 Sentence denotes Phylogenetic analysis
T28 4277-4683 Sentence denotes To 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).
T29 4684-4819 Sentence denotes Sequence alignment was performed using MUltiple Sequence Comparison by Log- Expectation (MUSCLE) software (http://www.clustal.org) [6].
T30 4820-5116 Sentence denotes 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].
T31 5117-5193 Sentence denotes Support for the tree topology was estimated with 1,000 bootstrap replicates.
T32 5194-5296 Sentence denotes The maximum likelihood phylogenetic tree in the Figure shows a main clade containing several clusters.
T33 5297-5540 Sentence denotes 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).
T34 5541-5664 Sentence denotes The latter was closely related to that of another sample taken from the same patient (GISAID accession ID: EPI_ISL_410545).
T35 5665-5848 Sentence denotes 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).
T36 5849-6033 Sentence denotes Figure 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)
T37 6034-6041 Sentence denotes GISAID:
T38 6042-6095 Sentence denotes Global Initiative on Sharing All Influenza Data; HKY:
T39 6096-6132 Sentence denotes Hasegawa, Kishino, and Yano; MEGA X:
T40 6133-6260 Sentence denotes Molecular Evolutionary Genetics Analysis across Computing Platforms; SARS-CoV-2: severe acute respiratory syndrome coronavirus.
T41 6261-6312 Sentence denotes Main clusters are highlighted in different colours.
T42 6313-6398 Sentence denotes The Wuhan reference genome is in larger font (GenBank accession number: NC_045512.2).
T43 6399-6523 Sentence denotes The filled circles represent the main supported clusters (bootstrap support values are indicated at the level of the nodes).
T44 6524-6610 Sentence denotes The scale bar at the bottom of the tree represents 0.000050 nt substitutions per site.
T45 6611-6757 Sentence denotes 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.
T46 6758-6954 Sentence denotes 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).
T47 6955-7173 Sentence denotes 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.
T48 7174-7269 Sentence denotes a The tree wasbuilt by using the best fitting substitution model (HKY) through MEGA X software.
T49 7270-7604 Sentence denotes 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).
T50 7605-7801 Sentence denotes In 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.
T51 7802-7923 Sentence denotes There was for example a cluster formed by two sequences from England and a cluster formed by three sequences from France.
T52 7924-8359 Sentence denotes Using 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.
T53 8360-8497 Sentence denotes The genome-wide SNPs are reported in Table 1 (positions referred respect to the reference sequence; GenBank accession number: NC_045512).
T54 8498-8593 Sentence denotes The corresponding amino-acid positions and variations inside the proteins are shown in Table 2.
T55 8594-8791 Sentence denotes Table 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)
T56 8792-8940 Sentence denotes SARS-CoV-2 sequence ID (country from which the sequence originated) 241 3037 10265 11083 13206 14408 15806 23403 26144 28881 28882 28883
T57 8941-9070 Sentence denotes 5' UTR ORF1ab gene ORF1ab gene ORF 1ab gene ORF1ab gene ORF1ab gene ORF1ab gene Gene S ORF3a gene Gene N Gene N Gene N
T58 9071-9124 Sentence denotes NC_045512 (China) C C G G C C A A G G G G
T59 9125-9177 Sentence denotes MN908947 (China) C C G G C C A A G G G G
T60 9178-9237 Sentence denotes EPI_ISL:412972 (Mexico) T T G G G T - G G A A C
T61 9238-9246 Sentence denotes EPI_ISL:
T62 9247-9299 Sentence denotes 412912 (Germany) T T A G C T A G G A A C
T63 9300-9308 Sentence denotes EPI_ISL:
T64 9309-9361 Sentence denotes 406862 (Germany) T T G G C C A G G G G G
T65 9362-9420 Sentence denotes EPI_ISL_412973 (Italy) T T G G C T A G G G G G
T66 9421-9479 Sentence denotes EPI_ISL_412974 (Italy) C C G T C C A A T G G G
T67 9480-9702 Sentence denotes N: 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.
T68 9703-9787 Sentence denotes a SNPs are shown according to nt positions in the genome sequence and gene location.
T69 9788-9896 Sentence denotes b The two sequences characterised in this study are the ones from Italy (EPI_ISL_412973 and EPI_ISL_412974).
T70 9897-10106 Sentence denotes Table 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)
T71 10107-10180 Sentence denotes SARS-CoV-2 strains 924 3334 3606 4314 4704 5170 614 251 203 204
T72 10181-10314 Sentence denotes ORF1ab ORF1ab ORF1ab ORF1ab ORF1ab ORF1ab Surface glycoprotein ORF3a Nucleocapsid phosphoprotein Nucleocapsid phosphoprotein
T73 10315-10362 Sentence denotes NC_045512 (China) F G L A P Q D G R G
T74 10363-10409 Sentence denotes MN908947 (China) F G L A P Q D G R G
T75 10410-10464 Sentence denotes EPI_ISL:412972 (Mexico) F G L G L -c G G K R
T76 10465-10473 Sentence denotes EPI_ISL:
T77 10474-10520 Sentence denotes 412912 (Germany) F S L A L Q G G K R
T78 10521-10529 Sentence denotes EPI_ISL:
T79 10530-10576 Sentence denotes 406862 (Germany) F G L A P Q G G R G
T80 10577-10629 Sentence denotes EPI_ISL_412973 (Italy) F G L A L Q G G R G
T81 10630-10682 Sentence denotes EPI_ISL_412974 (Italy) F G F A P Q D V R G
T82 10683-10800 Sentence denotes ORF: open reading frame; ORF1ab: ORF encoding polyprotein; SARS-CoV-2: severe acute respiratory syndrome coronavirus.
T83 10801-10975 Sentence denotes a 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.
T84 10976-11084 Sentence denotes b The two sequences characterised in this study are the ones from Italy (EPI_ISL_412973 and EPI_ISL_412974).
T85 11085-11116 Sentence denotes c -: possible sequencing error.
T86 11117-11364 Sentence denotes 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).
T87 11365-11492 Sentence denotes For the sequence of the Chinese tourist, the first SNP inside ORF1ab (bps 3037, AA 924) did not result in an amino acid change.
T88 11493-12026 Sentence denotes In 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.
T89 12027-12199 Sentence denotes The 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).
T90 12200-12307 Sentence denotes In ORF3a, this sequence had a V at amino acid position 251, as opposed to a G in the references from Wuhan.
T91 12308-12478 Sentence denotes Meanwhile, 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).
T92 12479-12664 Sentence denotes 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.
T93 12665-12836 Sentence denotes With 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 genomes.
T94 12838-12848 Sentence denotes Discussion
T95 12849-13109 Sentence denotes In this study, the full length genomes of two SARS-CoV-2 strains (EPI_ISL_412973 and EPI_ISL_412974) isolated in Italy, one from an Italian patient, the other from a Chinese tourist visiting Rome, are completely sequenced and analysed, after virus cultivation.
T96 13110-13287 Sentence denotes Compared to the viral genome sequence of the COVID-19 patient in Wuhan, the sequence from the Chinese tourist had two nt differences, while that of the Italian patient had four.
T97 13288-13407 Sentence denotes Phylogenetic analysis consistently placed the Italian patient’s strain in a distinct cluster from the tourist’s strain.
T98 13408-13659 Sentence denotes The strain of the Italian patient grouped with other viral strains identified in Germany and Mexico, while the strain from the Chinese tourist, related with the Wuhan virus strain, clustered with different European strains and a strain from Australia.
T99 13660-13876 Sentence denotes Other sequences from strains collected in Europe, which were included in the phylogenetic analysis, ended up in separate clusters from the ones respectively containing the sequences of the two patients reported here.
T100 13877-14080 Sentence denotes The results are consistent with several introductions of SARS-CoV-2 in Europe and/or further circulation of the single strain originating in Wuhan with concurrent evolution and accumulation of mutations.
T101 14081-14318 Sentence denotes The mutations found in the virus identified in Lombardy, compared with the reference Wuhan strain, and the identification of amino acids changes, should be further investigated to understand whether they may affect virus characteristics.
T102 14319-14560 Sentence denotes Some limitations need be mentioned: first, the lack of epidemiological information available with most sequences deposited in the database; second, the number of genomes available at the time of the analysis and consequently their selection.
T103 14561-14677 Sentence denotes Nevertheless, these data may be useful to understand the dynamics of the local transmission of SARS-CoV-2 in Europe.
T104 14679-14695 Sentence denotes Acknowledgements
T105 14696-14775 Sentence denotes We gratefully acknowledge the Collaborator Members of ISS COVID-19 study group:
T106 14776-15185 Sentence denotes Angela Di Martino, Marzia Facchini, Fabiola Mancini, Simona Puzelli, Giuseppina Di Mario, Laura Calzoletti, Stefano Fontana, Giulietta Venturi, Claudia Fortuna, Giulia Marsili, Antonello Amendola, Fabrizio Barbanti, Maria Scaturro, Daniela Fortini, Marco Di Luca, Luciano Toma, Francesco Severini, Patrizio Pezzotti, Antonino Bella, Flavia Riccardo, dept. of Infectious Diseases, Istituto Superiore di Sanità.
T107 15186-15365 Sentence denotes We gratefully acknowledge the Authors, the Originating and Submitting Laboratories for their sequence and metadata shared through GISAID and NCBI, on which this research is based.
T108 15366-15434 Sentence denotes All submitters of data may be contacted directly via www.gisaid.org.
T109 15435-15519 Sentence denotes The Acknowledgments Table for GISAID and NCBI is reported as Supplementary material.
T110 15520-15629 Sentence denotes The authors also thank Davide Mileto, Alessandro Mancon, Clinical Microbiology, Virology and Bioemergency, L.
T111 15630-15929 Sentence denotes Sacco University Hospital, Milan, Italy and Concetta Castilletti, Eleonora Lalle, Francesca Colavita, Fabrizio Carletti, Daniele Lapa, Licia Bordi Laboratory of Virology, National Institute for Infectious Diseases “Lazzaro Spallanzani” IRCCS, Rome, Italy, for performing diagnostic SARS-Cov-2 tests.
T112 15930-15941 Sentence denotes Disclaimer:
T113 15942-16036 Sentence denotes The new sequences have been deposited in GISAID with accession IDs: EPI_ISL_412973 and 412974.
T114 16038-16059 Sentence denotes Conflict of interest:
T115 16060-16074 Sentence denotes None declared.
T116 16075-16640 Sentence denotes Authors’ contributions: PS: conceived the study, coordinated the in vitro virus cultivation and wrote the manuscript together with FL and ALP; SFiore, EB,AM,CF prepared the Vero cells and cultivated the two virus; GF, AF, RDS and SFillo performed the whole genome sequence of the new genomes (ID: EPI_ISL_412973 and 412974); ALP, AA, AC analysed the genomes together with GF and FL; MRCapobianchi and MRG diagnosed SARS-CoV-2 onto the two samples; AC set up the molecular methods; MRCastrucci coordinated laboratory confirmation; GR read and revised the manuscript.
T117 16641-16686 Sentence denotes The authors read and approved the manuscript.
T118 16688-16706 Sentence denotes Supplementary Data
T119 16707-16729 Sentence denotes Supplementary Material
T120 16730-16766 Sentence denotes Click here for additional data file.