Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing

Abstract

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.

Differences in Genetic Diversity of Mammalian Tick-Borne Flaviviruses

“…A phylogenetic tree was constructed from the consensus genome sequences of the MTBFs sequenced in this study, a TBEV-Sib sequence from Genbank [note the authors do not have a TBEV-Sib isolate available for these studies], and a West Nile virus strain NY99 previously sequenced in the author’s laboratory, as an outgroup ( Figure 1 ). Overall, the MTBFs showed the expected relationship at the consensus genome level as reported in previous studies [ 9 , 11 , 27 , 28 , 29 ] with AHFV and KFDV closely related to each other (considered as genotypes), TBEV-FE, TBEV-Eur and OHFV closely related to each other, and POWV and DTV closely related to each other (considered as genotypes).…”

An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples

“…To overcome these difficulties, the targeted enrichment of viral genomes from clinical samples using PCR has been used to reduce the nonspecific reads, streamline the bioinformatics analysis, and allow greater accuracy compared with metagenomics [ 31 ]. Recent studies have demonstrated that amplicon-based approaches offer greater genome coverage and higher sensitivity than mNGS [ 40 , 41 , 42 , 43 ]. These protocols are designed to amplify short and overlapping amplicons (about 1000 bp or less) across the genome and have been used to sequence the Zika virus, Crimean–Congo hemorrhagic fever virus, and SARS-CoV-2 from clinical samples [ 40 , 41 , 44 , 45 ].…”

Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater

“…Given the advantages of NGS panels, they have also been evaluated and tested for the multiple detection and determination of various foodborne pathogens. Prior to the use of NGS panels, the detection and identification of foodborne pathogens was conducted using 16S rRNA sequencing based on Sanger sequencing, 16S rRNA-based metagenome and random genome sequencing-based shotgun metagenomics approaches ( Bridier, 2019 ); however, these detection methods produce an overabundance of sequence information ( Zakotnik et al, 2022 ). To overcome this problem, NGS panels were developed and evaluated using specific primer sets, generally targeting the virulence factors and antibiotic resistance genes of foodborne pathogens.…”