Patients, virus cultivation and whole genome sequencing
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. The Chinese patient had had onset of symptoms on 29 January 2020 and had been diagnosed in a hospital in Rome. The Italian patient whose onset of symptoms had occurred on 10 February 2020 had been diagnosed in a hospital in Milan. 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.
The samples used for this study were nasopharyngeal swabs. 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. 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. 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). 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. 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). 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]. 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/ ).