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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 dif...

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Autores principales: Zakotnik, Samo, Knap, Nataša, Bogovič, Petra, Zorec, Tomaž Mark, Poljak, Mario, Strle, Franc, Avšič-Županc, Tatjana, Korva, Miša
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9231111/
https://www.ncbi.nlm.nih.gov/pubmed/35746738
http://dx.doi.org/10.3390/v14061267
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author Zakotnik, Samo
Knap, Nataša
Bogovič, Petra
Zorec, Tomaž Mark
Poljak, Mario
Strle, Franc
Avšič-Županc, Tatjana
Korva, Miša
author_facet Zakotnik, Samo
Knap, Nataša
Bogovič, Petra
Zorec, Tomaž Mark
Poljak, Mario
Strle, Franc
Avšič-Županc, Tatjana
Korva, Miša
author_sort Zakotnik, Samo
collection PubMed
description 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.
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spelling pubmed-92311112022-06-25 Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing Zakotnik, Samo Knap, Nataša Bogovič, Petra Zorec, Tomaž Mark Poljak, Mario Strle, Franc Avšič-Županc, Tatjana Korva, Miša Viruses Article 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. MDPI 2022-06-10 /pmc/articles/PMC9231111/ /pubmed/35746738 http://dx.doi.org/10.3390/v14061267 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zakotnik, Samo
Knap, Nataša
Bogovič, Petra
Zorec, Tomaž Mark
Poljak, Mario
Strle, Franc
Avšič-Županc, Tatjana
Korva, Miša
Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing
title Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing
title_full Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing
title_fullStr Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing
title_full_unstemmed Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing
title_short Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing
title_sort complete genome sequencing of tick-borne encephalitis virus directly from clinical samples: comparison of shotgun metagenomic and targeted amplicon-based sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9231111/
https://www.ncbi.nlm.nih.gov/pubmed/35746738
http://dx.doi.org/10.3390/v14061267
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