20.500.12556/RUL-145240 Complete genome sequencing of tick-borne encephalitis virus directly from clinical samples comparison of shotgun metagenomic and targeted amplicon-based sequencing 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 (>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. tick-borne encephalitis virus whole genome sequencing metagenomics amplicons next-generation sequencing NGS clinical samples virus klopnega encefalitisa sekvenciranje celotnega genoma metagenomika true false true Angleški jezik Slovenski jezik Članek v reviji 2023-04-13 14:02:13 2023-04-13 14:02:18 2023-04-14 03:50:00 0000-00-00 00:00:00 2022 0 0 16 str. iss. 6, art. 1267 Vol. 14 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616.9 1999-4915 10.3390/v14061267 111682563 RAZ_Zakotnik_Samo_2022.pdf RAZ_Zakotnik_Samo_2022.pdf 1 D66B46C5D36F44D0F6FE2FF1C05FEC6A 491ca2c21c24c55bf8e597cb057cd777f2941cd10ccb93d2ee7f14e574ee5727 e3ab4e2d-d9f2-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167967 https://www.mdpi.com/1999-4915/14/6/1267 0 f8b3ba82-d9f2-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167968 Medicinska fakulteta 0 0 0