key: cord-287101-k3zq75zc authors: Micheli, V.; Rimoldi, S. G.; Romeri, F.; Comandatore, F.; Mancon, A.; Gigantiello, A.; Brilli, M.; Mileto, D.; Pagani, C.; Lombardi, A.; Gismondo, M. R.; Laboratory of Clinical Microbiology, Virology and Diagnostic of Bioemergencies Group title: Geographic reconstruction of the SARS-CoV-2 outbreak in Lombardy (Italy) during the early phase date: 2020-07-24 journal: nan DOI: 10.1101/2020.07.23.20159871 sha: doc_id: 287101 cord_uid: k3zq75zc The circulation of SARS-CoV-2 in Italy has been dominated by two large clusters of outbreaks in Northern part of the peninsula, source of alarming and prolonged infections in Lombardy region, in Codogno and Bergamo areas especially. The aim of the study was to expand understanding on the circulation of SARS-CoV-2 in the affected Lombardy areas. To this purpose, twenty full length genomes were collected from patients addressing to several Lombard hospitals from February 20th to April 4th, 2020. The obtained genome assemblies, available on the GISAD database and performed at the Referral Center for COVID-19 diagnosis, identified 2 main monophyletic clades, containing 9 and 52 isolates, respectively. The molecular clock analysis estimated a clusters divergence approximately one month before the first patient identification, supporting the hypothesis that different SARS-CoV-2 strains spread all over the world at different time, but their presence became evident only in late February along with Italian epidemic emergence. Therefore, the epidemiological reconstruction carried out by this work highlights multiple inputs of the virus into its initial circulation in Lombardy Region. However, a phylogenetic reconstruction robustness will be improved when other genomic sequences will be available, in order to guarantee a complete epidemiological surveillance. The positive-strand RNA virus family Coronaviridae includes 2 subfamilies for a total of 5 genera and more than 40 species, which infect a broad spectrum of vertebrates; 5 in particular, seven . 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 (which was not certified by peer review) The copyright holder for this preprint this version posted July 24, 2020. . coronaviruses are related to human diseases, among which two represented a public health concern in the past 20 years: in November 2002, the Severe Acute Respiratory Syndrome related coronavirus (SARS-CoV) emerged in Guangdong, China, resulting in more than 8,000 confirmed cases and 774 deaths in 37 countries; similarly, starting from 2012 in Saudi Arabia, the Middle East Respiratory Syndrome related coronavirus (MERS-CoV) infected 2,494 individuals and caused 858 fatalities. 6, 7 The COVID-19 related virus was classified as a β -betacoronavirus and, considering its close correlation to SARS-CoV, it was renamed SARS-CoV-2. 8 In Europe, Italy is one of the most affected areas, accounting for more than 230,000 cases on June 5 th , 2020. 9 Northern Italy has reported the highest prevalence in the country, especially in Milano, Brescia and Bergamo provinces, which registered more than 23,000, 14,000 and 13,000 cases 9 , The aim of the present study was to assess the potential presence of different viral clusters belonging to the six main provinces involved in Lombardy COVID-19 cases in order to highlight peculiar province-dependent viral characteristics. . 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 preprint this version posted July 24, 2020. . https://doi.org/10.1101/2020.07.23.20159871 doi: medRxiv preprint The study included SARS-CoV is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 24, 2020. For each sample, the genome assembly was obtained using a mapping-based approach, as follows. Low quality reads bases were trimmed out using Trimmomatic software, 10 is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 24, 2020. . https://doi.org/10.1101/2020.07.23.20159871 doi: medRxiv preprint the basis of the identified SNPs and the reference sequence. Reference bases were called in conserved positions with coverage above five, otherwise N were introduced. The procedure produced thirteen genome assemblies per sample, corresponding to the thirteen different trimming parameter sets. For each sample, the genome assembly with the lowest number of undetermined bases (N) as the final genome assembly was selected. A dataset of 41 genome assemblies of Sars-Cov-2 strains isolated in Italy between 20 February 2020 and 30 March 2020 were retrieved from GISAID database 12 , (see Supplementary Table 1 for details). A global dataset including these 41 GISAID (Table 1 ) genome assemblies and the 20 genome assemblies produced in this study was produced and aligned using MAFFT. 13 The low quality alignment regions at the extremities of the alignment were removed using Gblocks with default parameters. 14 The obtained alignment was subjected to Maximum Likelihood phylogenetic analysis using RaxML. 15 The obtained tree was then analysed using TempEst 16 19 Then the phylogenetic analysis was repeated with the selected coalescent priori with 100 million states and sampling every 1,000 steps. The convergence of MCMC chain was checked using Tracer v.1.7.1. 20 The maximum clade credibility (MCC) trees were obtained from the tree posterior distribution using TreeAnnotator (http://beast.community/index.html). . 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 preprint this version posted July 24, 2020. . A total of 20 samples from different subjects were successfully sequenced and included in phylogenetic analysis, attributing the progressive ID HSacco-N (from HSacco-2 to HSacco-21). All patients were resident in Lombardy Region, distributed in different provinces, as reported in table X: in particular, the category 'Milano' contains also patients hospitalized for non-COVID-19 disease, for whom SARS-CoV-2 hospital acquisition was supposed; in addition, HSacco-20, in 'Other' category, was a nurse living in Bergamo and working at Lodi Hospital. The obtained 20 Sars-Cov-2 genome assemblies are available on the GISAD database (see Table 2 for details). The comparison of the marginal likelihoods of constant and exponential coalescent models under a log-normal relaxed clock showed that the best fitting model was the exponential coalescent prior (PS BF exponential growth vs. constant = 2,42; SS BF exponential growth vs. constant = -2,39). The obtained phylogenetic tree is reported in Figure 1 . The tree shows the existence of two major monophyletic clades, containing 9 and 52 isolates respectively (coloured in green and blue, respectively, in Figure 1 ). The smaller clade is characterized by a non-synonymous mutation in position three of the gene M (mutation D3G) and its time of the Most Recent Common Ancestor (tRMCA) resulted 20 th February 2020 (HPD 95% interval from 4 th January to 24 th February); the Bergamo and HSacco-20 sequences mapped inside . 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 preprint this version posted July 24, 2020. . this group. The larger clade contains two monophyletic sub-clades, characterized by nonsynonymous mutations in the N gene ( Figure 1 ). The first cluster includes 19 isolates, mainly from Central Italy, in which R203K and G204R variants were found; the other one, instead, accounted for three sequences from Friuli Venezia Giulia region, presenting the V246I substitution. The tMRCA calculated for the two sub-clades were 2 nd March 2020 (HPD 95% interval from February 24 th to March 6 th ) and 28 th February 2020 (HPD 95% interval from February 20 th to March 2 nd ), respectively ( Table 2) . The Lombardy region has the highest prevalence of COVID-19 in Italy, thus being the likely epicentre of country outbreak. However, it is still unclear how SARS-CoV-2 circulation started: no epidemiological link was found for the first identified autochthonous patient. Moreover, despite the diagnosis was made on 20 February 2020, emerging evidences suggest a multiple virus introduction at least in January 2020. 21, 22, 23 The present work tried to expand understanding on the circulation of SARS-CoV-2 during the early epidemic period in Lombardy, especially in most affected areas. A phylogenetic analysis was conducted on 20 full length genomes collected from patients addressing to several Lombard hospitals from February 20 to April 4, 2020, creating a dataset including 41 Italian viral genome assemblies available on GISAID database as of 30th March, 2020. In spite of restricted geographical origin, these sequences were not included in the same cluster, but two main monophyletic clades containing isolates collected across different regions were found; in addition, the main clade accounting for 52/61 assemblies was divided in two subclades. Interestingly, the molecular clock analysis estimated a clusters divergence approximately one month before the first patient identification. Such evidences are in accordance with other studies, further supporting the . 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 preprint this version posted July 24, 2020. . https://doi.org/10.1101/2020.07.23.20159871 doi: medRxiv preprint hypothesis that different SARS-CoV-2 strains spread all over the world at different time, but their presence became evident only in late February along with Italian epidemic emergence. 21, 24, 25 Noteworthy, Bergamo (HSacco-5, HSacco-6, HSacco-11) and HSacco-20 sequences had the M gene D3G mutation of the first clade, in contrast with all the other ones located in the most represented clade. Two main observations consequently arose: firstly, the virus found in Bergamo area seemed to had a more restricted circulation, probably for a delayed introduction. In addition, the nurse working in Lodi and living in Bergamo likely acquired the infection in a personal contest rather than hospital environment, since Lodi patients mapped within the other cluster. Another interesting outcome was the presence of the N gene mutations R203K and G204R subclade. These two aminoacid changes appear to coexist, since they were always found together into the dataset, as well as they both are present almost exclusively in the GISAID 20B clade. These mutations have an impact on structure and function of the mutated N protein. The phylogenetic tree also clearly showed that the subclade is predominantly made up of isolates from Abruzzo region, suggesting a segregation of this specific virus in Central Italy area. Given that the role of these mutations is still unclear, an Indian group investigated their possible influence on virus replication interference mediated by miRNA: they found out how some miRNA, present in different pathological conditions, are likely to bind to native N gene and repress its expression, thus helping in disease progression limitation; on the contrary, mutated variants could increase their chances of interference escape. 26 Another small subclade was found in the main one, characterized by the presence of N gene mutation V246I in three sequences, all from Friuli Venezia Giulia region. Besides the unique geographical origin, it is noticeable that in GISAID map 'Geography' the V246I mutation is actually present only in Italy, as well as the V246A one only in Israel. Their rarity could have two probable explanations: on the one hand, available data are still limited, making difficult to have a reliable distribution; on the other hand, these variations could have a negative influence on viral fitness, diminishing efficacy in replication and consequently virus transmission. . 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) The copyright holder for this preprint this version posted July 24, 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) The copyright holder for this preprint this version posted July 24, 2020. . https://doi.org/10.1101/2020.07.23.20159871 doi: medRxiv preprint World Health Organization A pneumonia outbreak associated with a new coronavirus of probable bat origin World Health Organization; WHO Director-General's remarks at the media briefing on 2019-nCoV on World Health Organization; WHO Director-General's opening remarks at the media briefing on COVID-19 -11 International Committee on Taxonomy of Viruses; Virus Taxonomy Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding SARS and MERS: recent insights into emerging coronaviruses Coronaviridae Study Group of the International Committee on Taxonomy of Viruses; The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 Dipartimento della Protezione Civile; COVID-19 Italia -Monitoraggio della situazione Trimmomatic: a flexible trimmer for Illumina sequence data A framework for variation discovery and genotyping using next-generation DNA sequencing data disease and diplomacy: GISAID's innovative contribution to global health 2490-2492) Parallelization of MAFFT for large-scale multiple sequence alignments Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies Exploring the temporal structure of heterochronous sequences using TempEst jModelTest 2: more models, new heuristics and parallel computing Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10 Virus Evolution 4 Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology. syy032 A doubt of multiple introduction of SARS-CoV-2 in Italy: A preliminary overview Genomic characterization and phylogenetic analysis of SARS-COV-2 in Italy Emergence of genomic diversity and recurrent mutations in SARS-CoV-2 Mutations in SARS-CoV-2 viral RNA identified in Eastern India: Possible implications for the ongoing outbreak in India and impact on viral structure and host susceptibility Decoding SARS-CoV-2 Transmission and Evolution and Ramifications for COVID-19 Diagnosis Insights on early mutational events in SARSCoV-2 virus reveal founder effects across geographical regions Other mutations found in the present work had negligible influence on phylogenetic analysis, even a biological significance can not be excluded: viral genome and proteins are key factors in patients management and any variation can extremely burden the efficacy of drugs, vaccines and diagnostic tools or be related to a more severe clinical presentation. 27, 28 In conclusion, this study gave insights on early dynamics of SARS-CoV-2 circulation in Italy, underling peculiar strains localization and supporting multiple virus introductions at least in January 2020.