{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T13:18:01Z","timestamp":1774271881630,"version":"3.50.1"},"reference-count":26,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,21]],"date-time":"2026-03-21T00:00:00Z","timestamp":1774051200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NextGeneration EU-MUR PNRR Extended Partnership Initiative on Emerging Infectious Diseases INF-ACT","award":["PE00000007"],"award-info":[{"award-number":["PE00000007"]}]},{"name":"Ministero dell\u2019Universit\u00e0 e della Ricerca, Progetti di Rilevante Interesse Nazionale PRIN2022 PNRR","award":["P2022WYNAH"],"award-info":[{"award-number":["P2022WYNAH"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Toscana virus (TOSV) is an arthropod-borne virus, transmitted by sandflies, which represents a major cause of aseptic meningitis in Mediterranean countries during summer months. Despite its epidemiological importance, recent genomic data on circulating Italian strains remain limited. We performed comprehensive phylogenetic and genotypic characterization of 34 TOSV isolates (32 obtained from human biological samples and 2 from sandfly homogenates) collected between 2022 and 2025 from Northern\/Central Italy. All the sequenced isolates clustered within Lineage A, with strains circulating in Tuscany showing significantly lower intra group genetic divergence (p &lt; 0.05), indicative of compartmentalized local circulation. Both S and M segments exhibited negative selection and identified non-synonymous mutations deserving functional investigation. This study documents stable Lineage A predominance across Italian regions, with Tuscany showing distinct phylogeographic structuring. mNGS proves effective for TOSV genomic surveillance, supporting refined public health strategies, including targeted sandfly control in endemic foci.<\/jats:p>","DOI":"10.3390\/pathogens15030338","type":"journal-article","created":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T11:59:36Z","timestamp":1774267176000},"page":"338","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Molecular Epidemiology of Toscana Virus in Northern and Central Italy Using Metagenomic Next-Generation Sequencing"],"prefix":"10.3390","volume":"15","author":[{"given":"Giulia","family":"Alessandri","sequence":"first","affiliation":[{"name":"Virology Unit, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Giada","family":"Beligni","sequence":"additional","affiliation":[{"name":"Virology Unit, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2287-9258","authenticated-orcid":false,"given":"Gianni","family":"Gori Savellini","sequence":"additional","affiliation":[{"name":"Virology Unit, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8662-0782","authenticated-orcid":false,"given":"Alessandra","family":"Mistral De Pascali","sequence":"additional","affiliation":[{"name":"Department of Surgical and Neuronal Sciences, Alma Mater Studiorum, University of Bologna, 40138 Bologna, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9233-5737","authenticated-orcid":false,"given":"Federica","family":"Gobbo","sequence":"additional","affiliation":[{"name":"Laboratorio di Entomologia Sanitaria e Patogeni Trasmessi da Vettori, Istituto Zooprofilattico Sperimentale delle Venezie, Viale del Universit\u00e0 10, 35020 Legnaro, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5004-5763","authenticated-orcid":false,"given":"Fabrizio","family":"Montarsi","sequence":"additional","affiliation":[{"name":"Laboratorio di Entomologia Sanitaria e Patogeni Trasmessi da Vettori, Istituto Zooprofilattico Sperimentale delle Venezie, Viale del Universit\u00e0 10, 35020 Legnaro, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9414-0287","authenticated-orcid":false,"given":"Davide","family":"Mileto","sequence":"additional","affiliation":[{"name":"SC Microbiologica Clinica, Virologia e Diagnostica Bioemergenze, ASST Fatebenefratelli Sacco, Polo Universitario \u201cL. Sacco\u201d, Via G.B. Grassi 74, 20157 Milano, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Letizia","family":"Rizzo","sequence":"additional","affiliation":[{"name":"Virology Unit, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8869-8164","authenticated-orcid":false,"given":"Maria Grazia","family":"Cusi","sequence":"additional","affiliation":[{"name":"Virology Unit, Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy"},{"name":"Microbiology and Virology Unit, Santa Maria delle Scotte University Hospital, Viale Bracci 1, 53100 Siena, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,21]]},"reference":[{"key":"ref_1","first-page":"22","article-title":"Toscana virus epidemiology: From Italy to beyond","volume":"4","author":"Cusi","year":"2010","journal-title":"Open Virol. J."},{"key":"ref_2","first-page":"397","article-title":"Ecological and epidemiological studies of Toscana virus, an arbovirus isolated from Phlebotomus","volume":"18","author":"Verani","year":"1982","journal-title":"Ann. Ist. Super Sanita."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"104452","DOI":"10.1016\/j.jcv.2020.104452","article-title":"Epidemiology of Toscana virus in South Tuscany over the years 2011\u20132019","volume":"128","author":"Gandolfo","year":"2020","journal-title":"J. Clin. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"655","DOI":"10.1016\/j.cmi.2019.12.015","article-title":"An update on Toscana virus distribution, genetics, medical and diagnostic aspects","volume":"26","author":"Ayhan","year":"2020","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1016\/j.jinf.2013.11.006","article-title":"Toscana virus infections: A case series from France","volume":"68","author":"Dupouey","year":"2014","journal-title":"J. Infect."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"135","DOI":"10.5501\/wjv.v1.i5.135","article-title":"Emergence of Toscana virus in the Mediterranean area","volume":"1","author":"Charrel","year":"2012","journal-title":"World J. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Laroche, L., Ba\u00f1uls, A.L., Charrel, R., Fontaine, A., Ayhan, N., and Prudhomme, J. (2024). Sand flies and Toscana virus: Intra-vector infection dynamics and impact on Phlebotomus perniciosus life-history traits. PLoS Negl. Trop Dis., 18.","DOI":"10.1101\/2024.02.25.582017"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1701","DOI":"10.3201\/eid1111.050851","article-title":"Toscana virus in Spain","volume":"11","author":"Collao","year":"2005","journal-title":"Emerg. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1038\/s44298-025-00113-0","article-title":"Genetic diversity of Toscana virus glycoproteins affects the kinetics of virus entry and the infectivity of newly produced virions","volume":"3","author":"Thiesson","year":"2025","journal-title":"NPJ Viruses"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Ayhan, N., Prudhomme, J., Laroche, L., Ba\u00f1uls, A.-L., and Charrel, R.N. (2020). Broader geographical distribution of Toscana virus in the Mediterranean region suggests the existence of larger varieties of sand fly vectors. Microorganisms, 8.","DOI":"10.3390\/microorganisms8010114"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"105601","DOI":"10.1016\/j.meegid.2024.105601","article-title":"Fifty years after the first identification of Toscana virus in Italy: Genomic characterization of viral isolates within lineage A and aminoacidic markers of evolution","volume":"122","author":"Marsili","year":"2024","journal-title":"Infect. Genet. Evol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1128\/JCM.37.3.649-652.1999","article-title":"Laboratory diagnosis of Toscana virus infection by enzyme immunoassay with recombinant viral nucleoprotein","volume":"37","author":"Soldateschi","year":"1999","journal-title":"J. Clin. Microbiol."},{"key":"ref_13","first-page":"123","article-title":"Metagenomic next-generation sequencing in infectious diseases: Clinical applications, translational challenges, and future directions","volume":"14","author":"Xu","year":"2025","journal-title":"Infect. Dis. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6115","DOI":"10.2147\/IDR.S370964","article-title":"The comparison of metagenomic next-generation sequencing with conventional microbiological tests for identification of pathogens and antibiotic resistance genes in infectious diseases","volume":"15","author":"Lu","year":"2022","journal-title":"Infect. Drug Resist."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Wang, J., Han, Y., and Feng, J. (2019). Metagenomic next-generation sequencing for mixed pulmonary infection diagnosis. BMC Pulm. Med., 19.","DOI":"10.1186\/s12890-019-1022-4"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1111\/j.1863-2378.2011.01436.x","article-title":"Toscana Virus (TOSV) exposure is confirmed in blood donors from Central, North and South\/Southeast Anatolia, Turkey","volume":"59","author":"Ergunay","year":"2012","journal-title":"Zoonoses Public Health"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.tmaid.2012.11.006","article-title":"Seroprevalence of Toscana virus among residents of Aegean Sea islands, Greece","volume":"11","author":"Anagnostou","year":"2013","journal-title":"Travel Med. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1040.e1","DOI":"10.1016\/j.cmi.2015.07.012","article-title":"Virus isolation, genetic characterization and seroprevalence of Toscana virus in Algeria","volume":"21","author":"Alkan","year":"2015","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1186\/s12883-021-02528-7","article-title":"Toscana virus encephalitis in Southwest Germany: A retrospective study","volume":"21","author":"Dersch","year":"2021","journal-title":"BMC Neurol."},{"key":"ref_20","unstructured":"Istituto Superiore di Sanit\u00e0 (ISS) (2025). National Surveillance of Arboviral Diseases\u2014Toscana Virus, ISS. Available online: https:\/\/www.epicentro.iss.it\/arbovirosi\/archivio-2025."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1038\/s42003-025-08159-9","article-title":"Development of a novel amplicon based whole-genome sequencing framework for improved surveillance of Toscana virus","volume":"8","author":"Brandolini","year":"2025","journal-title":"Commun. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1038\/s41541-017-0021-9","article-title":"Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells","volume":"2","author":"Lokugamage","year":"2017","journal-title":"Npj Vaccines"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1007\/s12250-021-00384-w","article-title":"Genetic Mutation of SARS-CoV-2 during Consecutive Passages in Permissive Cells","volume":"36","author":"Chen","year":"2021","journal-title":"Virol. Sin."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1007\/s00705-025-06420-4","article-title":"Phenotypic and genetic changes of Crimean-Congo hemorrhagic fever virus during serial passage in susceptible cell lines","volume":"170","author":"Zsidei","year":"2025","journal-title":"Arch. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1186\/1743-422X-4-41","article-title":"Genome sequence analysis of La Crosse virus and in vitro and in vivo phenotypes","volume":"4","author":"Bennett","year":"2007","journal-title":"Virol. J."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"17097","DOI":"10.1038\/s41598-018-35472-7","article-title":"A single-cycle replicable Rift Valley fever phlebovirus vaccine carrying a mutated NSs confers full protection from lethal challenge in mice","volume":"8","author":"Terasaki","year":"2018","journal-title":"Sci. Rep."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/3\/338\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T12:25:49Z","timestamp":1774268749000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/3\/338"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,21]]},"references-count":26,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["pathogens15030338"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15030338","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,21]]}}}