key: cord-0682348-zqezpvj4 authors: Bartolini, B.; Rueca, M.; Gruber, C. E. M.; Messina, F.; Giombini, E.; Ippolito, G.; Capobianchi, M. R.; Di Caro, A. title: The newly introduced SARS-CoV-2 variant A222V is rapidly spreading in Lazio region, Italy date: 2020-11-30 journal: nan DOI: 10.1101/2020.11.28.20237016 sha: 0cd0d4b99b20ab11f280b06be349b506635ac4e8 doc_id: 682348 cord_uid: zqezpvj4 A new SARS-CoV-2 clade (GV) characterized by S substitution A222V, first reported from Spain in March, is rapidly spreading across Europe. To establish the A222V variant involvement in the infection rise in Italy, all GISAID sequences from Italy and those from our Laboratory (Lazio) in the period June-October were analysed. A222V, first recognized in August, represents 11.2% of sequences in this period, reaching 100% of autochthonous sequences in October, supporting increased GV circulation in Italy. increased GV circulation in Italy. Monitoring the emergence and spread of viral variants is a critical issue of virological surveillance, particularly in the present SARS-CoV-2 pandemic. In fact, it is essential to study the epidemiological dynamic and for the prompt recognition of mutations that may i. affect diagnostic recognition, ii. hamper the effectiveness of vaccines and iii. reduce the effects of therapeutic intervention [1, 2] . A new variant carrying the A222V substitution in the spike protein (S) was first observed in Spain in March 2020, and rapidly spread since June across Europe [3], seeding a new clade (GV), that is at present accounting for about 6.6% of all GISAID submitted genomes (https://www.gisaid.org/, accessed Nov 3, 2020). Until recently, only one GV sequence was reported to GISAID from Italy, occurring in Sardinia, collected in August 25. We regularly perform whole genome sequence analysis to support the surveillance service in Lazio region, Italy. To investigate if this new variant plays a role in the abrupt rise of infections that is currently affecting . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 30, 2020. ; https://doi.org/10.1101/2020.11.28.20237016 doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. our country, we describe the sequences obtained from samples collected in our Laboratory (Rome, Lazio), covering the period of June-October. A total of 57 whole genome sequences obtained in our laboratory from June 1st and October 31st were included in the analysis. More in details, we included sequences obtained for surveillance purpose in the period June-July (n=46), sequences from persons with known travel history, sampled in the second half of August, when the resurgence of the outbreak in Italy started (n=5) and sequences from the third week of October, to represent the presently circulating virus variants (n=6). AmpliSeq™ SARS-CoV-2 research panel on GeneStudio™ S5 Prime System (Thermofisher). Raw reads with mean quality Phred score >20 were selected and trimmed using Trimmomatic software v.0.36 [4] . SARS-CoV-2 genomes were assembled using reference-based assembly method, with BWA v.0.7.12 [5] and Samtools v.1.3.1 [6] . Assembled contigs were then verified using Geneious 2019.2.3. Besides the sequences obtained on our laboratory, we also included all the sequences of SARS-CoV-2 strains collected from Italy between June 1 st and October 31 st and retrieved from GISAID [7] , resulting in a total of 89 good quality sequences. These were clustered at 100% using CD-HIT v.4.6 software [8] , providing 57 unique sequences, that were included in the phylogenetic three. Maximum likelyhood phylogenetic analysis was performed with IQ-TREE v.1.6.12, using General Time Reversible with empirical base frequencies plus Gamma model (GTR+F+I) and 1000 replicates; Wuhan-Hu-1 strain was adopted as phylogenetic outgroup (NCBI accession number: Samples collection date, sample type, viral clade and travel history of patients, are reported in table 1.A phylogenetic tree (Figure 1 ) encompassing all the 57 unique SARS-CoV-2 sequences from Italy, collected from June 1 st , 2020 was built. As can be seen, the phylogenetic tree clearly shows the presence of one cluster of sequences epidemiologically correlated, belonging to the clade GR and occurred in June in Rome, Lazio, that will be described in details elsewhere. Nine sequences obtained in our laboratory, and one reported to GISAID from Sardinia by Laboratorio Biologia . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 30, 2020. Despite the small number of sequences so far reported from Italy, our data support a suddenly increased circulation of the GV clade in Italy. The A222V variant represents 11.2% of sequences in the period June-October 2020. In particular, the GV infections initially observed in Italy, occurring in August, were observed in Sardinia and travellers returning to Lazio region from Sardinia and Spain. On the contrary, all the GV sequences obtained in October were from patients without travel history, therefore presumably autochthonous. Taken together, these data suggest the introduction of clade GV in Italy in August, possibly in Sardinia and in other regions (including Lazio) by returning travellers, followed by extensive local spread that is still ongoing. It is not known at the moment whether other Italian regions are experiencing rising circulation of this new clade, and more data are necessary to better describe the virus dynamics and spread in our country. Although A222V mutation is located in a region of spike protein distant from the cell receptor binding site [3], an advantage in spreading efficiency cannot be excluded and needs further investigation, also in view of its location in a putative B cell epitope [9] that may affect immune recognition. This work was performed within the framework of the COVID-19 outbreak response and surveillance program, and has been approved by the INMI Ethical Committee ("Comitato Etico INMI Lazzaro Spallanzani IRCCS/Comitato Etico Unico Nazionale Covid-19"; issue n. 214/20-11-2020). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 30, 2020. ; https://doi.org/10.1101/2020.11.28.20237016 doi: medRxiv preprint Emergence of genomic diversity and recurrent mutations in SARS-CoV-2 Genomic Diversity and Hotspot Mutations in 30,983 SARS-CoV-2 Genomes: Moving Toward a Universal Vaccine for the "Confined Virus"? Pathogens I˜naki Comas, Fernando Gonz´alez Candelas, SeqCOVID-SPAIN consortium, et al. Emergence and spread of a SARS-CoV-2 variant through Trimmomatic: A flexible trimmer for Illumina sequence data Fast and accurate short read alignment with Burrows-Wheeler transform The Sequence Alignment/Map format and SAMtools disease and diplomacy: GISAID's innovative contribution to global health CD-HIT: accelerated for clustering the next generation sequencing data Mining of epitopes on spike protein of SARS-CoV-2 from COVID-19 patients We gratefully acknowledge the contributors of genome sequences of the newly emerging coronavirus, i.e. the Originating and Submitting Laboratories, for sharing their sequences and other metadata through the GISAID Initiative, on which this research is based. None declared. The sequences have been deposited in GISAID with accession IDs: EPI_ISL_609990, EPI_ISL_609989, EPI_ISL_609999, EPI_ISL_609998, EPI_ISL_609997, EPI_ISL_609996, EPI_ISL_609995, EPI_ISL_609994, EPI_ISL_609993, EPI_ISL_609992, EPI_ISL_609991 2020-10 Naso-pharyngeal swab Not known GV . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Clades are indicated on the side of the tree, following the GISAID nomenclature.All nodes with bootstrap values higher than 75 are highlighted with a black point.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted November 30, 2020. ; https://doi.org/10.1101/2020.11.28.20237016 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted November 30, 2020. ; https://doi.org/10.1101/2020.11.28.20237016 doi: medRxiv preprint