{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:54:27Z","timestamp":1792806867253,"version":"4.5.0"},"reference-count":60,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,6,10]],"date-time":"2022-06-10T00:00:00Z","timestamp":1654819200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, the Slovenian Research Agency","award":["P3-0083"],"award-info":[{"award-number":["P3-0083"]}],"id":[{"id":"https:\/\/ror.org\/05njb9z20","id-type":"ROR","asserted-by":"crossref"}]},{"name":"the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, the Slovenian Research Agency","award":["J3-2515"],"award-info":[{"award-number":["J3-2515"]}],"id":[{"id":"https:\/\/ror.org\/05njb9z20","id-type":"ROR","asserted-by":"crossref"}]},{"name":"the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, the Slovenian Research Agency","award":["871029"],"award-info":[{"award-number":["871029"]}],"id":[{"id":"https:\/\/ror.org\/05njb9z20","id-type":"ROR","asserted-by":"crossref"}]},{"name":"European Virus Archive (GLOBAL project)","award":["P3-0083"],"award-info":[{"award-number":["P3-0083"]}],"id":[{"id":"https:\/\/ror.org\/05qrk8753","id-type":"ROR","asserted-by":"crossref"}]},{"name":"European Virus Archive (GLOBAL project)","award":["J3-2515"],"award-info":[{"award-number":["J3-2515"]}],"id":[{"id":"https:\/\/ror.org\/05qrk8753","id-type":"ROR","asserted-by":"crossref"}]},{"name":"European Virus Archive (GLOBAL project)","award":["871029"],"award-info":[{"award-number":["871029"]}],"id":[{"id":"https:\/\/ror.org\/05qrk8753","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The clinical presentation of tick-borne encephalitis virus (TBEV) infection varies from asymptomatic to severe meningoencephalitis or meningoencephalomyelitis. The TBEV subtype has been suggested as one of the most important risk factors for disease severity, but TBEV genetic characterization is difficult. Infection is usually diagnosed in the post-viremic phase, and so relevant clinical samples of TBEV are extremely rare and, when present, are associated with low viral loads. To date, only two complete TBEV genomes sequenced directly from patient clinical samples are publicly available. The aim of this study was to develop novel protocols for the direct sequencing of the TBEV genome, enabling studies of viral genetic determinants that influence disease severity. We developed a novel oligonucleotide primer scheme for amplification of the complete TBEV genome. The primer set was tested on 21 clinical samples with various viral loads and collected over a 15-year period using the two most common sequencing platforms. The amplicon-based strategy was compared to direct shotgun sequencing. Using the novel primer set, we successfully obtained nearly complete TBEV genomes (&gt;90% of genome) from all clinical samples, including those with extremely low viral loads. Comparison of consensus sequences of the TBEV genome generated using the novel amplicon-based strategy and shotgun sequencing showed no difference. We conclude that the novel primer set is a powerful tool for future studies on genetic determinants of TBEV that influence disease severity and will lead to a better understanding of TBE pathogenesis.<\/jats:p>","DOI":"10.3390\/v14061267","type":"journal-article","created":{"date-parts":[[2022,6,13]],"date-time":"2022-06-13T06:31:59Z","timestamp":1655101919000},"page":"1267","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0478-2009","authenticated-orcid":false,"given":"Samo","family":"Zakotnik","sequence":"first","affiliation":[{"name":"Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nata\u0161a","family":"Knap","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petra","family":"Bogovi\u010d","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Ljubljana University Medical Center, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8191-0994","authenticated-orcid":false,"given":"Toma\u017e Mark","family":"Zorec","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3216-7564","authenticated-orcid":false,"given":"Mario","family":"Poljak","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4163-0503","authenticated-orcid":false,"given":"Franc","family":"Strle","sequence":"additional","affiliation":[{"name":"Department of Infectious Diseases, Ljubljana University Medical Center, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6243-0688","authenticated-orcid":false,"given":"Tatjana","family":"Av\u0161i\u010d-\u017dupanc","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1501-1004","authenticated-orcid":false,"given":"Mi\u0161a","family":"Korva","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1099\/0022-1317-80-1-179","article-title":"Sequence Analysis and Genetic Classification of Tick-Borne Encephalitis Viruses from Europe and Asia","volume":"80","author":"Ecker","year":"1999","journal-title":"J. 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