Complete genome sequencing of tick-borne encephalitis virus directly from clinical samples : comparison of shotgun metagenomic and targeted amplicon-based sequencing

Zakotnik, Samo (author), Knap, Nataša (author), Bogovič, Petra (author), Zorec, Tomaž Mark (author), Poljak, Mario (author), Strle, Franc (author), Avšič-Županc, Tatjana (author), Korva, Miša (author)

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.

• Language: English
• Keywords: tick-borne encephalitis virus, whole genome sequencing, metagenomics, amplicons, next-generation sequencing, NGS, clinical samples
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2022
• Number of pages: 16 str.
• Numbering: Vol. 14, iss. 6, art. 1267
• PID: 20.500.12556/RUL-145240
• UDC: 616.9
• ISSN on article: 1999-4915
• DOI: 10.3390/v14061267
• COBISS.SI-ID: 111682563
• Publication date in RUL: 13.04.2023

Record is a part of a journal

• Title: Viruses
• Shortened title: Viruses
• Publisher: MDPI
• ISSN: 1999-4915
• COBISS.SI-ID: 517597977

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: virus klopnega encefalitisa, sekvenciranje celotnega genoma, metagenomika