Author/Editor		Zakotnik, Samo; Knap, Nataša; Bogovič, Petra; Zorec, Tomaž Mark; Poljak, Mario; Strle, Franc; Avšič-Županc, Tatjana; Korva, Miša
Title		Complete genome sequencing of tick-borne encephalitis virus directly from clinical samples
Type		članek
Vol. and No.		Letnik 14, št. 6
Publication year		2022
Volume		str. 1-16
ISSN		1999-4915 - Viruses
Language		eng
Abstract		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.
Keywords		tick-borne encephalitis virus whole genome sequencing metagenomics virus klopnega encefalitisa sekvenciranje celotnega genoma metagenomika