Isolation and Full-Length Genome Characterization of SARS- CoV-2 from COVID-19 Cases in Northern Italy Danilo Licastro, a Sreejith Rajasekharan, b Simeone Dal Monego, a Ludovica Segat, c Pierlanfranco D’Agaro, c Alessandro Marcello, b The Regione FVG Laboratory Group on COVID-19 a ARGO Open Lab Platform for Genome Sequencing, Trieste, Italy b Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy c Laboratorio di riferimento per SARS-CoV-2, Regione Friuli-Venezia Giulia, Azienda Sanitaria Universitaria Integrata Giuliano Isontina (ASUGI), UCO Igiene e Sanità Pubblica, Dipartimento di Scienze Mediche Chirurgiche e della Salute, Università di Trieste, Trieste, Italy Danilo Licastro and Sreejith Rajasekharan contributed equally to this work and are named in alphabetical order. KEYWORDS COVID-19, Italy, SARS-CoV-2 I n December 2019, the novel coronavirus severe acute respiratory syndrome corona- virus 2 (SARS-CoV-2) emerged in the city of Wuhan in Hubei province, People’s Republic of China, as the etiologic agent of coronavirus disease 2019 (COVID-19), which has hence spread worldwide causing a global pandemic (1–3). The epidemic has been growing exponentially in Italy for the last month, affecting over 60,000 individuals so far and with a heavy mortality burden. Italy is only anticipating what will be the trend in the whole of Europe and elsewhere. At the beginning of March 2020, the first nasopharyngeal swabs positive for SARS-CoV-2 started to be detected in the Northern Eastern Region of Friuli-Venezia Giulia. These identifications followed the expansion of the two clusters in Lombardy and Veneto that emerged in the previous weeks in northern Italy (4). Swab contents were seeded on Vero E6 cells and monitored for cytopathic effect and by an RT-PCR protocol using primers for the N region (5). Cell culture supernatants from passage 1 (P1) of four isolates were collected, and RNA was extracted with QIAamp viral RNA minikit (Qiagen) and quantified with an in vitro- transcribed RNA standard (S. Rajasekharan and A. Marcello, unpublished data). The quantity and quality of the RNA were assessed using Qubit 2.0 fluorometer (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies). For each sample, 100 ng of total RNA was processed using Zymo-Seq RiboFree ribosomal depletion library preparation kit (Zymo Research). All the obtained libraries passed quality check and were quantified before being pooled at equimolar concentration and sequenced on Illumina Nano MiSeq 2- by 150-bp paired-end mode following standard procedures. Sequenced reads that passed the quality check (Phred score 30) were adaptor and quality trimmed, and the remaining reads were assembled de novo using Megahit (v.1.2.9) with default parameter settings. Megahit generated in all cases 7 contigs with more than 1,000 bp and 100 coverage; all of these assembled contigs were compared (using BLASTn) against the entire nonredundant (nr) nucleotide and protein databases. In all cases the longest and more covered contigs were identified as MT019532.1, “Severe acute respiratory syndrome coronavirus 2 isolate BetaCoV/Wuhan/IPBCAMS- WH-04/2019, complete genome,” with 99% identity and 0 gaps. The longer sequences were named hCoV-19/Italy/FVG/ICGEB_S1, _S5, _S8, and _S9 and were deposited in GISAID (see below). Sequence analysis showed an uneven coverage along the SARS- CoV-2 genome, with an average range from 126 to 7,576 reads and a mean coverage per sample of 1,169 (Fig. 1). Phylogenetic trees were inferred using the maximum likelihood method implemented in the MEGAX program using the GISAID sequences Citation Licastro D, Rajasekharan S, Dal Monego S, Segat L, D’Agaro P, Marcello A, The Regione FVG Laboratory Group on COVID-19. 2020. Isolation and full-length genome characterization of SARS-CoV-2 from COVID-19 cases in northern Italy. J Virol 94:e00543-20. https://doi.org/10.1128/JVI.00543-20. Editor Rozanne M. Sandri-Goldin, University of California, Irvine Copyright © 2020 American Society for Microbiology. All Rights Reserved. Address correspondence to Pierlanfranco D’Agaro, pdagaro@units.it, or Alessandro Marcello, marcello@icgeb.org. Accepted manuscript posted online 1 April 2020 Published LETTER TO THE EDITOR crossm June 2020 Volume 94 Issue 11 e00543-20 jvi.asm.org 1 Journal of Virology 18 May 2020 Downloaded from https://journals.asm.org/journal/jvi on 28 November 2021 by 3.238.127.182.