Molecular Tracing of SARS-CoV-2 in Italy in the First Three Months of the Epidemic

  1. Alessia Lai
  2. Annalisa Bergna
  3. Sara Caucci
  4. Nicola Clementi
  5. Ilaria Vicenti
  6. Filippo Dragoni
  7. Anna Cattelan
  8. Stefano Menzo
  9. Angelo Pan
  10. Annapaola Callegaro
  11. Adriano Tagliabracci
  12. Arnaldo Caruso
  13. Francesca Caccuri
  14. Silvia Ronchiadin
  15. Claudia Balotta
  16. Maurizio Zazzi
  17. Emanuela Vaccher
  18. Massimo Clementi
  19. Massimo Galli
  20. Gianguglielmo Zehender

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Abstract

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly-characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

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  1. Version published to 10.3390/v12080798
  2. Version published to 10.20944/preprints202007.0144.v1
  3. ScreenIT

    SciScore for 10.1101/2020.07.06.20147140: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Eighteen sequences were obtained after isolating the virus in Vero E6 cells while the remaining 41 were obtained directly from biological samples such as nasopharyngeal swabs or broncho-alveolar lavages (39 and 2, respectively).
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The results were mapped and aligned to the reference genome obtained from GISAID (https://www.gisaid.org/, accession ID: EPI_ISL_412973) using Geneious software, v. 9.1.5 (http://www.geneious.com) [6] or Torrent Suite v.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    5.10.1 or BWA-mem and rescued using Samtools alignment/Map (v 1.9).
    BWA-mem
    suggested: (Sniffles, RRID:SCR_017619)
    Samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Alignment was performed using MAFFT [7] and manually cropped to a final length of 29,779 bp using BioEdit v.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    BioEdit
    suggested: (BioEdit, RRID:SCR_007361)
    2.3 Genetic distance, recombination and selection pressure analyses: The MEGA X program was used to evaluate the genetic distance between and within Italian sequences on the full length genome, with variance estimation performed using 1,000 bootstrap replicates [8].
    MEGA X
    suggested: None
    All of the genes were tested for selection pressure using Datamonkey (https://www.datamonkey.org/). 2.4 Phylogenetic and phylodynamic analyses: The simplest evolutionary model best fitting the sequence data was selected using the JmodelTest v.
    Datamonkey
    suggested: (DataMonkey, RRID:SCR_010278)
    JmodelTest
    suggested: (jModelTest, RRID:SCR_015244)
    The phylogenetic analysis for clade assignment was performed by RaxML [11] on the entire dataset of 588 genomes.
    RaxML
    suggested: (RAxML, RRID:SCR_006086)
    A root-to-tip regression analysis was made using TempEst in order to investigate the temporal signal of the dataset [15].
    TempEst
    suggested: (TempEst, RRID:SCR_017304)
    The final trees were summarised by selecting the tree with the maximum product of posterior probabilities (pp) (maximum clade credibility or MCC) after a 10% burn-in using Tree Annotator v.1.10.4 (included in the BEAST package), and were visualised using FigTree v.
    BEAST
    suggested: (BEAST, RRID:SCR_010228)
    FigTree
    suggested: (FigTree, RRID:SCR_008515)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a protocol registration statement.

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  4. Version published to 10.1101/2020.07.06.20147140v1 on medRxiv