Expedited retrieval of high-quality Usutu virus genomes via Nanopore sequencing with and without target enrichment

Abstract

Usutu virus (USUV) is a mosquito-borne zoonotic virus and one of the causes of flavivirus encephalitis in birds and occasionally in humans. USUV rapidly disperses in a susceptible host and vector environment, as is the case in South and Central Europe. However, compared to other flaviviruses, USUV has received less research attention and there is therefore limited access to whole-genome sequences and also to in-depth phylogenetic and phylodynamic analyses. To ease future molecular studies, this study compares first- (partial sequencing via Sanger), second- (Illumina), and third-generation (MinION Nanopore) sequencing platforms for USUV. With emphasis on MinION Nanopore sequencing, cDNA-direct and target-enrichment (amplicon-based) sequencing approaches were validated in parallel. The study was based on four samples from succumbed birds commonly collected throughout Germany. The samples were isolated from various sample matrices, organs as well as blood cruor, and included three different USUV lineages. We concluded that depending on the focus of a research project, amplicon-based MinION Nanopore sequencing can be an ideal cost- and time-effective alternative to Illumina in producing optimal genome coverage. It can be implemented for an array of lab- or field-based objectives, including among others: phylodynamic studies and the analysis of viral quasispecies.

Reconstruction of the molecular evolution of Usutu virus in Germany: Insights into virus emersion and circulation

“…Regular and stringent genomic surveillance of viral pathogens supports outbreak investigations by providing evidence for their transmission routes and geographic spread. Since only a few whole genome sequences of USUV from Germany are available (67, 47, 8, 83, 33, 15, 49), a better phylogenetic analysis is the next essential step in understanding the spread of USUV in Germany as well as in Europe. A preferred method using third-generation sequencing is a nanopore sequencing approach based on target-enrichment through amplicon generation (84, 67).…”

“…Since only a few whole genome sequences of USUV from Germany are available (67, 47, 8, 83, 33, 15, 49), a better phylogenetic analysis is the next essential step in understanding the spread of USUV in Germany as well as in Europe. A preferred method using third-generation sequencing is a nanopore sequencing approach based on target-enrichment through amplicon generation (84, 67). This recently established protocol (67) was used to gain insight into the distribution and expansion of USUV throughout five consecutive years in Germany.…”

“…For WGS, USUV RNA-positive samples were selected primarily on the basis of their geographic location in order to represent a comprehensive picture of Germany. The cycle threshold (Ct) values and to some extent also the lineages, as already determined by partial sequencing (46, 45, 67), were used as further decision criteria. Throughout all five years, USUV isolates from the two different bird orders Passeriformes and Strigiformes were identified and sequenced.…”

“…Abbreviation: CNS: central nervous system; BB: Berlin Brandenburg; BE: Berlin; BW: Baden-Württemberg; BY: Bavaria; HE: Hesse; NI: Lower Saxony; MV: Mecklenburg-West Pomerania; NW: North Rhine-Westphalia; SL: Saarland; SN: Saxony; ST: Saxony-Anhalt; TH: Thuringia Nanopore sequencing and data analysis Nanopore sequencing was performed as described by Holicki et al 2022 (67). In short, RNA was reverse transcribed using the SuperScript IV First-Strand cDNA Synthesis Reaction Kit (Cat.…”

Vaccination of Zoo Birds against West Nile Virus—A Field Study

“…All sampled zoo birds were screened for the presence of WNV and USUV by RT-qPCR. In case of a positive WNV-RT-qPCR result, whole genome sequencing was performed using MinION by Oxford Nanopore-technology (Oxford Science Park, the United Kingdom) similar to that previously described for USUV [ 51 ]. In short, complementary DNA (cDNA) was synthesised by multiplex PCR using SuperScript IV First-Strand cDNA Synthesis Reaction (Cat.…”