Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin
Introduction: Toscana virus (TOSV, Phlebovirus genus) is a neurotropic sandfly-transmitted virus whose distribution encompasses various countries within the Mediterranean basin. TOSV infection remains a non-notifiable disease in Europe with no official surveillance plans in place despite being consi...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-03-01
|
| Series: | International Journal of Infectious Diseases |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1201971224007513 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850241077129248768 |
|---|---|
| author | Miss Martina Brandolini Mr Giorgio Dirani Ms Silvia Zannoli Dr Alessandra Mistral De Pascali Ms Ludovica Ingletto Ms Laura Dionisi Ms Claudia Colosimo Prof Monica Cricca Prof Alessandra Scagliarini Prof Vittorio Sambri |
| author_facet | Miss Martina Brandolini Mr Giorgio Dirani Ms Silvia Zannoli Dr Alessandra Mistral De Pascali Ms Ludovica Ingletto Ms Laura Dionisi Ms Claudia Colosimo Prof Monica Cricca Prof Alessandra Scagliarini Prof Vittorio Sambri |
| author_sort | Miss Martina Brandolini |
| collection | DOAJ |
| description | Introduction: Toscana virus (TOSV, Phlebovirus genus) is a neurotropic sandfly-transmitted virus whose distribution encompasses various countries within the Mediterranean basin. TOSV infection remains a non-notifiable disease in Europe with no official surveillance plans in place despite being considered a significant public health concern in Southern Mediterranean countries. In order to mitigate the impact of this virus on human health, it is crucial to gain a deeper understanding beyond the anecdotal and fragmented evidence that currently exists. The aim of this study is to develop an amplicon-based whole genome sequencing method suitable for the genomic characterization of TOSV. Methods: Primers were designed on TOSV lineage A S, M and L segments reference sequences (GenBank: GCA_031497085.1) using Primal Scheme web tool to generate 400 bp tiled amplicons. To guarantee coverage and sensitivity, a multi-sequence alignment of GenBank-available complete sequences was used to identify mismatches and introduce degenerations in relevant ambiguous sites. Two-pool multiplex amplicon-based library preparation was carried out with Illumina Microbial Amplicon Prep (iMAP, Illumina) and custom designed primers. Two viral isolates on Vero E6 cells and three clinical samples of patients presenting with meningitis were sequenced. De novo assembly was performed with BaseSpace DRAGEN Targeted Microbial (version 1.3.1). Results: A set of 45 oligonucleotide primer pairs (26 for segment L, 13 for segment M and 6 for segment S) that amplify overlapping segments spanning TOSV genome was generated. The primers sets were subsequently compared to an alignment of 8 sequences for segment L, 16 for segment M and 32 for segment S to introduce degenerations. Genome sequencing yielded a mean 98.2% coverage for high-viral-titre propagates on Vero E6 cell culture. Two human samples (1 urine and 1 cerebrospinal fluid) with Ct (Cycle threshold) values of 32 and 33 yielded to sequences with coverage of 91.3% and 80.3%. Another urine sample with Ct = 35 yielded to 47% of mean coverage. Discussion: Our study, albeit with preliminary and limited but very promising results for propagates and samples with relatively high Ct values, proposes a new whole genome amplicon-based sequencing method for genomic characterization of TOSV one of the most neglected and least monitored arboviruses. Alongside an increasingly necessary systematic epidemiological monitoring of TOSV infections, WGS may be instrumental to unravel its genomic makeup and to gain insights into its biology and transmission dynamics. Conclusion: Taking advantage and making the most of the sequencing infrastructures, bioinformatics pipelines, and data-sharing platforms, established during the COVID-19 pandemic, it will make possible to establish an integrated surveillance led by sentinel territorial network to targeting TOSV and other endemic and emerging Arboviruses. This knowledge can inform the development of new diagnostics, therapeutics, and preventive measures, particularly in TOSV high risk regions. |
| format | Article |
| id | doaj-art-43bdca185c844f5789f8c0bbf835e47f |
| institution | OA Journals |
| issn | 1201-9712 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | International Journal of Infectious Diseases |
| spelling | doaj-art-43bdca185c844f5789f8c0bbf835e47f2025-08-20T02:00:42ZengElsevierInternational Journal of Infectious Diseases1201-97122025-03-0115210767610.1016/j.ijid.2024.107676Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean BasinMiss Martina Brandolini0Mr Giorgio Dirani1Ms Silvia Zannoli2Dr Alessandra Mistral De Pascali3Ms Ludovica Ingletto4Ms Laura Dionisi5Ms Claudia Colosimo6Prof Monica Cricca7Prof Alessandra Scagliarini8Prof Vittorio Sambri9Department of Medical and Surgical Sciences, University of BolognaUnit of Microbiology, The Greater Romagna Area Hub Laboratory, AUSL RomagnaUnit of Microbiology, The Greater Romagna Area Hub Laboratory, AUSL RomagnaDepartment of Medical and Surgical Sciences, University of BolognaDepartment of Medical and Surgical Sciences, University of BolognaDepartment of Medical and Surgical Sciences, University of BolognaDepartment of Medical and Surgical Sciences, University of BolognaDepartment of Medical and Surgical Sciences, University of BolognaDepartment of Medical and Surgical Sciences, University of BolognaDepartment of Medical and Surgical Sciences, University of BolognaIntroduction: Toscana virus (TOSV, Phlebovirus genus) is a neurotropic sandfly-transmitted virus whose distribution encompasses various countries within the Mediterranean basin. TOSV infection remains a non-notifiable disease in Europe with no official surveillance plans in place despite being considered a significant public health concern in Southern Mediterranean countries. In order to mitigate the impact of this virus on human health, it is crucial to gain a deeper understanding beyond the anecdotal and fragmented evidence that currently exists. The aim of this study is to develop an amplicon-based whole genome sequencing method suitable for the genomic characterization of TOSV. Methods: Primers were designed on TOSV lineage A S, M and L segments reference sequences (GenBank: GCA_031497085.1) using Primal Scheme web tool to generate 400 bp tiled amplicons. To guarantee coverage and sensitivity, a multi-sequence alignment of GenBank-available complete sequences was used to identify mismatches and introduce degenerations in relevant ambiguous sites. Two-pool multiplex amplicon-based library preparation was carried out with Illumina Microbial Amplicon Prep (iMAP, Illumina) and custom designed primers. Two viral isolates on Vero E6 cells and three clinical samples of patients presenting with meningitis were sequenced. De novo assembly was performed with BaseSpace DRAGEN Targeted Microbial (version 1.3.1). Results: A set of 45 oligonucleotide primer pairs (26 for segment L, 13 for segment M and 6 for segment S) that amplify overlapping segments spanning TOSV genome was generated. The primers sets were subsequently compared to an alignment of 8 sequences for segment L, 16 for segment M and 32 for segment S to introduce degenerations. Genome sequencing yielded a mean 98.2% coverage for high-viral-titre propagates on Vero E6 cell culture. Two human samples (1 urine and 1 cerebrospinal fluid) with Ct (Cycle threshold) values of 32 and 33 yielded to sequences with coverage of 91.3% and 80.3%. Another urine sample with Ct = 35 yielded to 47% of mean coverage. Discussion: Our study, albeit with preliminary and limited but very promising results for propagates and samples with relatively high Ct values, proposes a new whole genome amplicon-based sequencing method for genomic characterization of TOSV one of the most neglected and least monitored arboviruses. Alongside an increasingly necessary systematic epidemiological monitoring of TOSV infections, WGS may be instrumental to unravel its genomic makeup and to gain insights into its biology and transmission dynamics. Conclusion: Taking advantage and making the most of the sequencing infrastructures, bioinformatics pipelines, and data-sharing platforms, established during the COVID-19 pandemic, it will make possible to establish an integrated surveillance led by sentinel territorial network to targeting TOSV and other endemic and emerging Arboviruses. This knowledge can inform the development of new diagnostics, therapeutics, and preventive measures, particularly in TOSV high risk regions.http://www.sciencedirect.com/science/article/pii/S1201971224007513 |
| spellingShingle | Miss Martina Brandolini Mr Giorgio Dirani Ms Silvia Zannoli Dr Alessandra Mistral De Pascali Ms Ludovica Ingletto Ms Laura Dionisi Ms Claudia Colosimo Prof Monica Cricca Prof Alessandra Scagliarini Prof Vittorio Sambri Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin International Journal of Infectious Diseases |
| title | Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin |
| title_full | Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin |
| title_fullStr | Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin |
| title_full_unstemmed | Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin |
| title_short | Novel Amplicon-Based Whole Genome Sequencing for Toscana Virus – A Breakthrough in Genome Analysis of a Neglected Virus in the Mediterranean Basin |
| title_sort | novel amplicon based whole genome sequencing for toscana virus a breakthrough in genome analysis of a neglected virus in the mediterranean basin |
| url | http://www.sciencedirect.com/science/article/pii/S1201971224007513 |
| work_keys_str_mv | AT missmartinabrandolini novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT mrgiorgiodirani novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT mssilviazannoli novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT dralessandramistraldepascali novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT msludovicaingletto novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT mslauradionisi novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT msclaudiacolosimo novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT profmonicacricca novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT profalessandrascagliarini novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin AT profvittoriosambri novelampliconbasedwholegenomesequencingfortoscanavirusabreakthroughingenomeanalysisofaneglectedvirusinthemediterraneanbasin |