Genomic characterization and phylogenetic analysis of SARS‐COV‐2 in Italy

Abstract This report describes the isolation, molecular characterization, and phylogenetic analysis of the first three complete genomes of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) isolated from three patients involved in the first outbreak of COVID‐19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS‐CoV‐2 was present in Italy weeks before the first reported cases of infection.


| INTRODUCTION
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a new coronavirus that causes severe respiratory diseases and is closely related to SARS-CoV, was described for the first time in the city of Wuhan in the Hubei province of China in late December 2019 (https://www.who.int/ csr/don/05-january-2020-pneumonia-of-unkown-cause-china/en/). It belongs to the β-coronavirus genus of the Coronaviridae family, and has 96% genomic identity with a previously detected SARS-like bat coronavirus. 1,2 The virus subsequently spread and, on 30 January 2020, the World Health Organisation (WHO) declared it a public health emergency of international concern (https://www.who.int/ news-room/detail/30-01-2020-statement-on-the-second-meeting-of-theinternational-health-regulations-(2005)-emergency-committee-regardingthe-outbreak-of-novel-coronavirus-(2019-nCoV). On 26 February, the Director-General of the WHO announced that the number of new cases of the disease, now officially known as COVID-19, reported outside China since the day before had for the first time exceeded the number of new cases in China (https://www.who.int/dg/speeches/detail/whodirector-general-s-opening-remarks-at-the-mission-briefing-oncovid-19---26-february-2020). By 13 March 2020, a total of 137 445 cases and 5088 fatalities had been reported in 117 countries (https:// gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda75947 40fd40299423467b48e9ecf6), giving rise to major concerns throughout the world, but particularly in South Korea, Iran, and Italy. We have now molecularly characterized and phylogenetically analyzed three complete genomes of SARS-CoV-2 isolated from Gianguglielmo Zehender and Alessia Lai contributed equally to this paper. three of the first 16 patients observed in Italy, none of whom reported a recent history of foreign travel.

| PATIENTS AND METHODS
All of the data used in this study were previously anonymized as using Geneious software, v. 9.1.5 (http://www.geneious.com). 4 The genomes obtained from the three patients were aligned with a total of 157 SARS-CoV-2 genomes obtained worldwide and publicly available at GISAID on 3 March 2020 (https://www.gisaid.org/), and with an additional Italian strain that became available during the study. Table S1 shows the accession IDs, and sampling dates and locations of the sequences included in the dataset.
A root-to-tip regression analysis was made using TempEst in order to investigate the temporal signal of the dataset. Two coalescent priors (constant population size and exponential growth) and strict vs relaxed molecular clock models were tested by means of path sampling (PS) and stepping stone (SS) sampling. 8 The substitution rate prior was set as a normal distribution with mean 2.2 × 10 −6 substitutions/site/day, standard deviation = 1.1 × 10 −6 (http:// virological.org/t/phylodynamic-analysis-176-genomes-6-mar-2020/356). 9 The time of the most recent common ancestor (tMRCA) was calculated using days as the unit of time.
All of the genes were tested for selection pressure using Datamonkey (https://www.datamonkey.org/).

| DISCUSSION
The present phylogenetic analysis confirms that the common origin of the SARS-CoV-2 strains characterized so far was several weeks before the first cases of COVID-19 pneumonia were described in China. 9 It also shows that the whole genomes of the three SARS-CoV-2 strains isolated from patients in northern Italy and characterized by us are closely related to each other, as well as to the other two published Italian sequences, and the German, Finnish, Mexican, and Brazilian sequences, all of which formed a highly supported clade.
The German sequence at the outgroup of the clade came from a COVID-19 outbreak reported between 20 and 24 January and occurring after business meetings with a Shanghai business woman who tested positive after returning to China. 10 Our tMRCA estimate showed that the root of clade A was in the month of January 2020, a period compatible with this event. However, our data do not allow us to make any hypotheses concerning the possible routes followed by the virus to reach Italy because, given the limited number of sparsely sampled sequences in the tree, 11 it is impossible to infer the directionality of transmission, and this means that multiple independent importations to Europe cannot be excluded.
Our data suggest that SARS-CoV-2 virus entered northern Italy between the second half of January and early February 2020, which is weeks before the first Italian case of COVID-19 was identified and therefore long before the current containment measures were taken.
F I G U R E 1 Root-to-tip regression analysis of the 161 SARS-CoV-2 sequences aligned F I G U R E 2 Dated tree of 161 SARS-CoV-2 sequences showing statistically significant support for clades along the branches (posterior probability >0.7). Clade A containing the Italian strains is highlighted in red. The patients characterized in this study are indicated by a symbol. The table shows the time of the most recent common ancestor (tMRCA) estimates and 95% high posterior density of the significant clade A nodes. SARS-CoV-2, severe acute respiratory syndrome coronavirus 2