Amplicon-Based MinION Sequencing Complements Severe Fever With Thrombocytopenia Syndrome (SFTS) Diagnosis via Real-Time RT-PCR in Patients With Suspected SFTS

Abstract

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal threat. Increasing Severe fever with thrombocytopenia syndrome (SFTS) risk in Asia and the United States stems from the spread of natural host, Haemaphysalis longicornis. Rapid and accurate SFTSV molecular diagnosis is crucial for treatment decisions, reducing fatality risk.

Methods: Blood samples from 17 suspected SFTS patients at Chuncheon Sacred Heart Hospital (September-December 2022) were collected. SFTSV was diagnosed using two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays from Gangwon Institute of Health and Environment (RT-qPCR/GIHE) and Asan Medical Center (RT-qPCR/AMC). To address RT-qPCR disparities, amplicon-based MinION sequencing traced SFTSV genomic sequences in clinical samples.

Results: In two samples (N39 and N50), SFTSV was detected in both RT-qPCR/GIHE and RT-qPCR/AMC. Among 11 samples, RT-qPCR/AMC exclusively detected SFTSV. In four samples, both assays yielded negative results. Amplicon-based MinION sequencing enabled nearly whole-genome sequencing of SFTSV in samples N39 and N50. Among 11 discordant samples, five contained significant SFTSV reads, aligning with the RT-qPCR/AMC findings. However, another six samples showed insufficient viral reads in accordance with the negativity observed in RT-qPCR/GIHE. The phylogenetic pattern of SFTSV demonstrated N39 formed a genetic lineage with genotype A in all segments. SFTSV N50 grouped with the B-1 sub-genotype for L segment and B-2 sub-genotype for the M and S segments, indicating genetic reassortment.

Conclusion: The study demonstrates the robust sensitivity of amplicon-based MinION sequencing for the direct detection of SFTSV in clinical samples containing ultralow copies of viral genomes. Next-generation sequencing holds potential in resolving SFTSV diagnosis discrepancies, enhancing understanding of diagnostic capacity, and risk assessment for emerging SFTSV.

Keywords: Amplicon-Based MinION Sequencing; Genomic Characteristics; Molecular Diagnosis; RT-qPCR; SFTSV.

Fig. 1. Phylogenetic analysis of severe fever with thrombocytopenia syndrome virus from patients with SFTS in Chuncheon, 2022. The phylogenetic trees were generated using maximum likelihood methods in MEGAX with bootstrap 1,000 iterations based on the ORF regions of the SFTSV (A) L (60–6,271 nt), (B) M (19–3,240 nt), and (C) S (70–1,703 nt) segments. The scale bars indicate the number of nucleotide substitutions per site. The numbers at each node are bootstrap probabilities determined for 1,000 replicates. The SFTSV obtained in this study is shown in red. The genetic clades indicate six genotypes (A–F) and three sub-genotypes (B1 to B-3) in the right panel.

SFTS = severe fever with thrombocytopenia syndrome, ORF = open reading frame, SFTSV = severe fever with thrombocytopenia syndrome virus.

Fig. 2. Tanglegram comparing the phylogenies between each segment of severe fever with thrombocytopenia syndrome virus in South Korea. Tanglegram comparing phylogenies between each segment of SFTSVs tripartite genomes (A) L–M segments, (B) L–S segments, and (C) M–S segments. The tanglegram was generated using the R package, using consensus maximum likelihood topologies based on the nucleotide sequences of each SFTSV genome. Letters for taxa are indicated in red for the SFTSV genome found in this study. The tanglegrams illustrate putative reassortment events by highlighting when two segments from the same viral isolate appear in different configurations between their respective ML trees.

SFTSV = severe fever with thrombocytopenia syndrome virus, ML = maximum-likelihood.