Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
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 |
_version_ | 1784735249958174720 |
---|---|
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. |
format | Online Article Text |
id | pubmed-9231111 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT zakotniksamo completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT knapnatasa completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT bogovicpetra completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT zorectomazmark completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT poljakmario completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT strlefranc completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT avsiczupanctatjana completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing AT korvamisa completegenomesequencingoftickborneencephalitisvirusdirectlyfromclinicalsamplescomparisonofshotgunmetagenomicandtargetedampliconbasedsequencing |