https://orcid.org/0000-0003-3178-8569
Figures
Abstract
Background
Whole-genome sequencing plays a critical role in the genomic epidemiology intended to improve understanding the spread of emerging viruses. Dabie bandavirus, causing severe fever with thrombocytopenia syndrome (SFTS), is a zoonotic tick-borne virus that poses a significant public health threat. We aimed to evaluate a novel amplicon-based nanopore sequencing tool to obtain whole-genome sequences of Dabie bandavirus, also known as SFTS virus (SFTSV), and investigate the molecular prevalence in wild ticks, Republic of Korea (ROK).
Principal findings
A total of 6,593 ticks were collected from Gyeonggi and Gangwon Provinces, ROK in 2019 and 2020. Quantitative polymerase chain reaction revealed the presence of SFSTV RNA in three Haemaphysalis longicornis ticks. Two SFTSV strains were isolated from H. longicornis captured from Pocheon and Cheorwon. Multiplex polymerase chain reaction-based nanopore sequencing provided nearly full-length tripartite genome sequences of SFTSV within one hour running. Phylogenetic and reassortment analyses were performed to infer evolutionary relationships among SFTSVs. Phylogenetic analysis grouped SFTSV Hl19-31-4 and Hl19-31-13 from Pocheon with sub-genotype B-1 in all segments. SFTSV Hl20-8 was found to be a genomic organization compatible with B-1 (for L segment) and B-2 (for M and S segments) sub-genotypes, indicating a natural reassortment between sub-genotypes.
Conclusion/Significance
Amplicon-based next-generation sequencing is a robust tool for whole-genome sequencing of SFTSV using the nanopore platform. The molecular prevalence and geographical distribution of SFTSV enhanced the phylogeographic map at high resolution for sophisticated prevention of emerging SFTS in endemic areas. Our findings provide important insights into the rapid whole-genome sequencing and genetic diversity for the genome-based diagnosis of SFTSV in the endemic outbreak.
Author summary
Next-generation sequencing (NGS) is important in understanding the genetic diversity, genomic epidemiology, and transmission chain of emerging viruses of concern. Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic vector-borne infectious disease with a high mortality rate. Approximately 170 SFTS cases are reported annually in Republic of Korea. We assessed a multiplex polymerase chain reaction-based NGS method for whole-genome sequencing of SFTS virus (SFTSV) and monitored the prevalence of SFTSV in wild ticks. We developed an amplicon-based NGS method to obtain whole-genome sequences of SFTSV using the nanopore sequencing. A total of 6,593 ticks were collected from Gyeonggi and Gangwon Provinces in 2019 and 2020. The infection and minimum infection rates of SFSTV were 0.34% and 0.05%, respectively. We recovered whole-genome sequences of SFTSV from three Haemaphysalis longicornis ticks collected from Pocheon and Cheorwon, enhancing the high-resolution phylogeographical analysis for potential prevention of SFTS outbreaks. Phylogenetic analysis showed that SFTSV Hl20-8 from Cheorwon was a reassortant compatible with the B-1 and B-2 sub-genotypes. Our findings provide important insights into the target enrichment-based NGS and genetic diversity for the genome-based diagnosis of SFTSV in the endemic outbreak.
Citation: Lee J, Park K, Kim J, Lee S-H, Lee G-Y, Cho S, et al. (2022) Whole-genome sequencing and genetic diversity of severe fever with thrombocytopenia syndrome virus using multiplex PCR-based nanopore sequencing, Republic of Korea. PLoS Negl Trop Dis 16(9): e0010763. https://doi.org/10.1371/journal.pntd.0010763
Editor: Paul O. Mireji, Kenya Agricultural and Livestock Research Organization, KENYA
Received: January 22, 2022; Accepted: August 23, 2022; Published: September 12, 2022
Copyright: © 2022 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All genomic sequences of SFTSV are available from the GenBank (accession numbers: M160952-OM160963 and OM160979-OM160984).
Funding: This study was supported by the Agency for Defense Development (UE202026GD to J.-W.S.) and the Institute for Basic Science Korea (IBS-R801-D92022-a03 to J.-W.S.). In addition, this research was supported by Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2021R1I1A2049607 to W.-K.K.). Partial funding was provided by the Armed Forces Health Surveillance Branch, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS); Silver Spring, MD (ProMIS ID #P0025-2016-2018-ME to T.A.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Severe fever with thrombocytopenia syndrome (SFTS) is a zoonotic vector-borne infectious disease with clinical symptoms including acute fever above 38°C, thrombocytopenia, leukopenia, and multiple organ dysfunction [1]. SFTS was first reported in China, followed by several reports from many other countries including Republic of Korea (ROK), Japan, Vietnam, and Taiwan [2–6]. Although the mean case mortality rate has varied among countries and with time, the average fatality rate of SFTS has remained relatively high in China (5.3–16.2%), ROK (23.3%), and Japan (27%) [7–9]. According to Korea Disease Control and Prevention Agency, approximately 170 SFTS patients have been recorded annually in the ROK [10]. There are no effective vaccines and antiviral therapeutics for SFTS despite the significant morbidity and mortality of the disease.
Dabie bandavirus (formerly called SFTS virus, SFTSV) is an enveloped negative-sense single-stranded RNA virus, belongs to the family Phenuiviridae, order Bunyavirales, [11]. The SFTSV genome contains three segments, including the large (L) segment, which encodes an RNA-dependent RNA polymerase; a medium (M) segment, which encodes two surface glycoproteins Gn and Gc; a small (S) segment, which encodes a nucleoprotein (NP) and a nonstructural S segment (NS) protein, respectively [12]. The life cycle and transmission mechanisms of SFTSV in nature remain unclear, although transmission via arthropod vectors is considered the most credible route, similar to that for other members of Phenuiviridae [13]. The Asian longhorned tick (Haemaphysalis longicornis) is the main transmission vector of SFTSV in East Asian countries [14]. SFTSV RNA has also been detected in several other tick species, including H. flava, Rhipicephalus microplus, Amblyomma testudinarium, Dermacentor nuttalli, Hyalomma asiaticum, and Ixodes nipponensis in endemic areas [15–18]. Furthermore, human-to-human transmission by contact with blood or body fluid from SFTS patients has been reported in ROK and China [19,20].
Next-generation sequencing (NGS) plays a critical role in understanding the genetic diversity, molecular epidemiology, and transmission chain of virus outbreaks [21–23]. Several NGS approaches, including sequence-independent single-primer amplification, small RNA deep sequencing, and target enrichment methods have been utilized for whole-genome sequencing (WGS) of emerging viruses with low viral copy numbers [24–27]. The MinION system (Oxford Nanopore Technologies, London, UK) is a portable device for real-time sequencing in field situations or hospitals [28,29]. Multiplex polymerase chain reaction (PCR)-based nanopore sequencing has been used to obtain nearly complete genome sequences of Hantaan virus from natural reservoir hosts to define the phylogeographical association and molecular evolution in the ROK [30]. Amplicon-based NGS methods with a nanopore system were developed to acquire full-length genomic sequences of severe acute respiratory syndrome coronavirus 2 for phylogenetic and epidemiological analyses [31]. However, to our knowledge, the amplicon-based NGS is yet to be performed for complete genome sequencing of SFTSV.
Methods
Ethics statement
This study was approved by the Korea University Institutional Animal Care and Use Committee (KU-IACUC) and performed with strict accordance to the recommendations of the KU-IACUC (No. #2019–171) guideline. All experiments were conducted in an animal biosafety level 3 (ABSL3) laboratory at Korea University.
Tick collection and species identification
Tick sampling (n = 6,593) was performed in Gyeonggi and Gangwon Provinces, ROK, during 2019–2020 (Fig 1). Ticks were collected by well-trained researchers (US Forces Korea and the ROKA) from military bases located in seven areas: Gyeonggi (Gapyeong, Paju, Pocheon, Pyeongtaek, and Yeoncheon) and Gangwon (Cheorwon and Inje) Provinces. Ticks were captured using the dragging method described in a previous study [32]. All samples were identified by morphological characterization and life stages, and subsequently transported to ABSL3 facility at Korea University [33,34]. The captured ticks were identified as H. longicornis (n = 5,607, 85.04%), H. flava (n = 309, 4.69%), H. phasiana (n = 1, 0.02%), I. nipponensis (n = 666, 10.10%), and I. persulcatus (n = 10, 0.15%) (Table 1). Among the life cycle stages of ticks, nymphs accounted for most of all ticks (n = 5,709, 86.59%), followed by larvae (n = 407, 6.17%), males (n = 264, 4.00%), and females (n = 213, 3.23%) (Table 2).
The red circles indicate the severe fever with thrombocytopenia syndrome virus (SFTSV) RNA positive sites: a, Pocheon in Gyeonggi Province; b, Cheorwon in Gangwon Province. The black circles show the regions where no SFTSV RNA was detected: c, Gapyeong; d, Paju; e, Pyeongtaek; and f, Yeoncheon in Gyeonggi Province; g, Inje in Gangwon Province. The geographic map was created by a Quantum Geographical Information System 3.10 software for Mac and modified using Adobe Illustrator CC 2019.
RNA extraction and cDNA synthesis
Tick specimens (larvae and nymphs, n = 1–30; adult, n = 1) were homogenized with stainless steel beads in 700 μL phosphate buffered saline using a FastPrep-24 5G benchtop homogenizer (MP Biomedicals, Irvine, CA, USA). Homogenates were centrifuged for 5 min at 8,600 × g and 4°C. The recovered supernatants were stored at −80°C until use. Total RNA was extracted from 250 μL aliquots of the supernatants using TRIzol LS Reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA) with SFTSV-PHS (5′-ACA CAR AGA CSS CC-3′).
Quantitative polymerase chain reaction (qPCR)
qPCR was performed from the prepared cDNA using SYBR Green PCR Master Mix (Applied Biosystems) on a Quantstudio 5 Flex Real-Time PCR System (Applied Biosystems). The reaction mixture contained 5 μL of 2X SYBR Green PCR Master Mix, 1 μL of 5 μM each primer (final concentration, 0.5 μM), 3 μL of nuclease-free water, and 1 μL cDNA template in a total volume of 10 μL. The forward and reverse primer sequences are SFTS-L-F (5’-ACC TCT TTG ACC CTG AGT TWG ACA-3’) and SFTS-L-R (5’-CTG AAG GAG ACA GGT GGA GAT GA-3’), respectively. The cycling conditions consisted of denaturation at 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. The cutoff Ct value was 40 in the assay.
Reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR was performed from total RNA of inoculated supernatants and cells to validate the isolation of SFTSV. Total RNA was extracted using TRIzol LS Reagent (Ambion) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems) with SFTSV-PHS. The reaction mixture contained 2.5 μL of 10X reaction buffer (JMR Holdings, London, UK), 200 μM dNTP (Elpis Biotech, Daejeon, Korea), 0.25 U of SuperTherm Taq polymerase (JMR Holdings), 10 μM each primer, and 1.5 μL of cDNA template in a total volume of 25 μL. SFTSV-specific primer sequences were SFTSV-L1F (outer and inner): 5’-ACA CAG AGA CGC CCA GAT-3’, SFTSV-L343R (outer): 5’-CAT CCC ATC AGA ACC ATC AT-3’, and SFTSV-L263R (inner): 5’-TGA AGT CAT GAT TGA TCT-3’ for the L segment [35]; SFTSV-M482F (outer): 5’-TCT GCA GTT CAG ACT CAG GGA-3’, SFTSV-M1242R (outer): 5’-GAC GTG TAT TGC TGT TTT CCC-3’, SFTSV-M549F (inner): 5’-TGT TGC TTG TCA GCC TAT GAC-3’, and SFTSV-M1222R (inner): 5’-CAA CCA ATG ATC CTG AGT GGA-3’ for the M segment [12]; SFTSV-S1123F (outer): 5’- TAA ACT TCT GTC TTG CTG GCT CC-3’, SFTSV-S1589R (outer and inner): 5’-ATC AAG AAG CTG AAG GAG ACA G-3’, and SFTSV-S1169F (inner): 5’-TGG TGA AGG CAT CTT GCC A-3’for the S segment [36]. The cycling conditions for first and nested PCR consisted of initial denaturation at 95°C for 5 min, followed by 6 cycles of denaturation at 95°C for 40 s, annealing at 37°C for 40 s, elongation at 72°C for 1 min, followed by 32 cycles of denaturation at 95°C for 40 s, annealing at 42°C for 40 s, elongation at 72°C for 1 min and then final elongation at 72°C for 7 min using ProFlex PCR System (Life Technology, Carlsbad, USA).
Cell lines
Vero E6 cells (ATCC, #DR-L2785) were purchased from ATCC. The cell lines were seeded and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% HEPES buffer, 1% L-glutamine (Lonza, Basel, Switzerland), and 0.1% gentamicin (Gibco, Life Technologies, Carlsbad, CA, USA). The plates were incubated at 37°C with 5% CO2 in an incubator.
Virus isolation
The aliquots of SFTSV-positive supernatant were inoculated into the prepared Vero E6 cells. After 90 min of adsorption, the excess inoculum was discarded, and the viral suspension was replaced with 5.5 mL of DMEM containing 5% FBS, 1% HEPES buffer, 1% L-glutamine, and 0.1% gentamicin. The cells were incubated at 37°C with 5% CO2 in an incubator and passaged at 7–10 days intervals.
Plaque assay
2×106 Vero E6 cells were seeded in the each well of 6-well plates. After overnight incubation at 37°C with 5% CO2, the each well was washed twice with PBS and inoculated with 10-fold serially diluted SFTSV. After 90 min absorption at 37°C, the cells were overlaid with the overlay medium and medium-melting-point agarose mix (2:1 ratio). The plaques were incubated at 37°C for 5 days and then visualized by staining the cells with 5% neutral red solution (Sigma-Aldrich, Burlington, USA).
Multiplex PCR
Multiplex PCR primers were designed for WGS of SFTSV L, M, and S segments. cDNA was amplified using SFTSV-specific primer mixtures and Solg 2X Uh-Taq PCR Smart mix (Solgent, Daejeon, ROK) according to the manufacturer’s instructions. The composition of 25 μL of the reaction mixture was 12.5 μL of 2× Uh pre-mix, 1.0 μL cDNA template, 10.0 μL of 0.5 μM each primer mixture (final concentration, 0.2 μM), and 1.5 μL of distilled water. The first PCR cycling was performed with the following cycling conditions: initial denaturation at 95°C for 15 min, 40 cycles at 95°C for 20 s, 50°C for 40 s, 72°C for 1 min, and final elongation at 72°C for 3 min. The second PCR was conducted in a 25 μL reaction mixture containing 12.5 μL 2× Uh pre-mix, 1.0 μL of the first PCR product, 10.0 μL of 0.5 μM each primer mixture (final concentration, 0.2 μM), and 1.5 μL of distilled water. The cycling conditions included an initial denaturation at 95°C for 15 min, followed by 25 cycles at 95°C for 20 s, 50°C for 40 s, 72°C for 1 min, and final elongation at 72°C for 3 min. The primer sequences are shown in S1 Table.
Nanopore sequencing
The DNA library was prepared using a Ligation Sequencing Kit (SQK-LSK109) with a Native Barcoding Kit (EXP-NBD104; Oxford Nanopore Technologies) according to the manufacturer’s instructions. The libraries were barcoded, pooled, and ligated to sequencing adapters. Purified libraries were loaded onto FLO-MIN106 (R9.4; Oxford Nanopore Technologies) and sequenced using the MinION device (Oxford Nanopore Technologies). Basecalling was performed by Guppy (v3.0.3) embedded in the MinIT system (Oxford Nanopore Technologies). Raw data were demultiplexed and the adaptor sequences were trimmed using MinKNOW software (Oxford Nanopore Technologies). The filtered reads were assembled into a single file using Porechop v.9.0. Viral reads were mapped to the reference genome sequences of SFTSV SPL114A, and consensus sequences were extracted by CLC Genomics Workbench (v7.5.2; Qiagen, Hilden, Germany). Manual polishing was performed using the indel error-correction method described previously [30].
Illumina sequencing
DNA libraries were prepared using TruSeq Nano DNA low-throughput sample preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. DNA templates were mechanically sheared by an M220 focused ultrasonicator (Covaris, Woburn, MA, USA). The fragmented amplicons were size-selected, A-tailed, ligated with indexes and adapters, and enriched by PCR. The quality and concentration of libraries were evaluated using the DNA 1000 chip kit (Agilent Technologies, Santa Clara, CA, USA) on a bioanalyzer (Agilent Technologies). The libraries were quantified by qPCR using Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA) on a Quantstudio 5 Flex Real-Time PCR System (Applied Biosystems). The pooled libraries were sequenced on a MiSeq benchtop sequencer (Illumina) with 2 × 150 bp using a MiSeq reagent kit v2 (Illumina). Adapter and index sequences were trimmed from the raw data and filtered reads were mapped to the reference genomic sequences of SFTSV SPL114A by CLC Genomics Workbench (v7.5.2; Qiagen). The consensus sequences were extracted from analyzed viral reads.
Rapid amplification of cDNA ends (RACE)
PCR was performed using 3′ and 5′ RACE System for Rapid Amplification of cDNA Ends version 2.0 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The primer sequences are shown in S2 Table.
Phylogenetic analysis
The tripartite genomic sequences of SFTSV were aligned using the Clustal W method in Lasergene version 5 (DNASTAR, Madison, WI, USA). Phylogenies were generated using the best fit GTR+G+I (for all segments) substitution models of evolution by the maximum likelihood method in MEGA7 [37]. The topologies were assessed by bootstrap analysis for 1,000 iterations.
Genetic reassortment analysis
Graph incompatibility-based reassortment finder (GiRaF) analysis was performed to estimate genetic reassortment events [38]. Alignments of SFTSV tripartite genomes were used as an input source for Bayesian analysis [39]. The optimal evolutionary models were determined using MEGA7. A total of 1,000 unrooted candidate trees were generated using GTR+G+I substitution model sampled every 200 iterations with a 25% burn-in. The analysis was repeated ten times with each independent MrBayes-based tree data. The default value of a confidence threshold was 0.7 for the data set.
Results
Epidemiological surveillance of SFTSV
Tick samples were pooled by criteria of the life cycle stage. If larvae and nymphs were captured at identical areas, they were pooled up to 30 individuals. A total of three pooled tick samples (Hl19-31-4, Hl19-31-13, and Hl20-8) were found to harbor SFTSV RNA. SFTSV Hl19-31-4 and Hl19-31-13 were collected from Pocheon in Gyeonggi Province, in 2019. SFTSV Hl20-8 was identified from Cheorwon in Gangwon Province, in 2020. Infection and minimum infection rates were 0.34% (3/886) and 0.05% (3/6,582), respectively (Table 3).
Virus isolation of SFTSV
Partial genomic sequences of SFTSV were identified from inoculated supernatants and cells (S1 Fig). The first plaques of SFTSV Hl19-31-4 and Hl20-8 were confirmed at 5 days post-inoculation, and the number of infectious particles was 1.2×106 and 2.0×106 PFU/mL, respectively (S2 Fig).
Multiplex PCR-based NGS of SFTSV using nanopore and Illumina sequencing
The workflow overview of multiplex PCR-based NGS for whole-genome sequencing of SFTSV is shown in Fig 2. Using multiplex PCR-based nanopore sequencing, nearly whole-genome sequences of SFTSV were recovered from the collected tick samples collected from Gyeonggi and Gangwon Provinces, ROK. The mean genome coverages were 99.43% for the L segment, 99.47% for the M segment, and 99.20% for the S segment, with over 50× depth of coverage at all regions for each segment after one hour of nanopore sequencing (Fig 3 and S3 Table). Average viral reads and depth for SFTSV tripartite genomes were determined using the mapped reads corresponding to sequencing running times.
Total RNA was extracted from tick samples and cDNA was synthesized with SFTSV-specific oligonucleotide primer (SFTSV-PHS). cDNA was enriched using designed SFTSV-specific primer mixtures for the next-generation sequencing. Amplified libraries were pooled, ligated to sequencing adapter, and sequenced using nanopore and Illumina sequencing according to each manufacturer’s instructions. Raw data were filtered and analyzed by CLC Genomics Workbench (v7.5.2).
The average depth of coverages of SFTSV tripartite genomes was shown by using amplicon-based nanopore sequencing at 5 min, 15 min, 30 min, and 1 h. The colors represent the coverage fold; 0 coverage (red), from 1 to 10 coverage (yellow), from 11 to 30 coverage (light green), from 31 to 50 and over 50 (green).
The tick specimens were sequenced using the Illumina MiSeq system to obtain whole-genome sequences of SFTSV. The coverage rates of SFTSV were 99.43% for the L segment, 99.47% for the M segment, and 99.20% for the S segment (S4 Table). The 3′ and 5′ terminal sequences of SFTSV L, M, and S segments were determined using RACE PCR.
Genetic diversity and genome exchange of SFTSV using the phylogenetic inference
The phylogenetic analysis showed that Hl19-31-4 and Hl19-31-13 from Pocheon were clustered with sub-genotype B-1 in all segments of SFTSV (Fig 4). The phylogenetic patterns of SFTSV L, M, and S segments demonstrated that Hl20-8 from Cheorwon had differing levels of incongruence in the phylogenies. The L segment of SFTSV Hl20-8 shared common ancestors with sub-genotype B-1, whereas M and S segments formed a clade with sub-genotype B-2.
The phylogenetic trees were generated using maximum likelihood methods in MEGA7 with bootstrap 1,000 iterations based on the complete ORF regions of the SFTSV (A) L (17–6,270 nt), (B) M (19–3,240 nt), and (C) S (29–1,702 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 lettering. The genetic clades indicate six genotypes (A-F) and three sub-genotypes (B-1 to B-3) in the right panel. The genomic sequences of SFTSV in this figure are described in S5 Table.
The occurrence of genetic reassortment of SFTSV was estimated using the GiRaF software (Fig 5). The genome exchanges were detected in SFTSV Hl20-8 between B-1 and B-2 sub-genotypes with over 0.9 confidence levels. The genome composition of SFTSV Hl20-8 was compatible with the B-1 sub-genotype (for the L segment) and the B-2 sub-genotype (for the M and S segments).
(A) The tripartite genomes of SFTSV were described using a pairwise distance model estimated using the graph incompatibility-based reassortment finder software. Phylogenetic trees were generated using the maximum likelihood method in MEGA7 with 1,000 bootstrap iterations based on the ORFs of the SFTSV (B) L (17–6,270 nt), (C) M (19–3,240 nt), and (D) S (29–1,702 nt) segments. SFTSVs are color-coded according to sub-genotype classification as follows: yellow, B-1; green, B-2; blue, B-3. Newly recovered SFTSV genomes are shown in red. The genomic sequences of SFTSV in this figure are described in S5 Table.
Discussion
High-throughput sequencing technologies have become robust tools for improving approaches to point-of-care diagnostics and molecular epidemiology of emerging diseases when used to track sources of epidemic infections [40–42]. NGS-based genomic surveillance with MinION system has been applied to elucidate virus characterization and transmission dynamics of Ebola virus, Zika virus, and severe acute respiratory syndrome coronavirus 2 in the field [43–45]. The extension of SFTSV genome sequences has been limited by the lack of a relevant sequencing assay with designed primers for attaining complete genomic sequences. We established a multiplex PCR-based NGS that generated the full-length genomic sequence of SFTSV from ticks using the primer set specific for SFTSV L, M, and S segments. The multiplex primers were designed from reference sequences deposited in NCBI, enabling whole-genome sequencing of all genotypes of SFTSV. Given the genetic diversity among variants of SFTSV, further studies are needed to determine the sensitivity of the assay on clinical and tick samples containing varied genotypes and ultra-low viral RNA copy numbers.
The spread of SFTSV is attributed to the geographical distribution of arthropod vectors and contacts between humans and infected tick populations [13]. Approximately 170 SFTS cases occur annually in ROK, affecting both military personnel and civilians [10]. Yoo et al. described the epidemiological association between molecular prevalence of SFTSV and tick populations with ecological environments in endemic areas [46]. Epidemiological surveillance has been performed to clarify the serological and molecular prevalence of SFTSV and the distribution of their reservoirs in the ROK [47–49]. Phylogeographic analysis of SFTSV elucidated an epidemiological association between viral sequences from ROK Army soldier with SFTS and wild ticks collected from the putative infection site [35]. The additional genome sequences of SFTSV with geographical information enable to define the phylogenetic and spatial relationships of SFTS patients with infectious sources at high-resolution [50,51]. In this study, whole-genome sequences of SFTSV were newly obtained from ticks (H. longicornis) collected from Pocheon and Cheorwon, enhancing the resolution of a phylogeographic map for the sophisticated prevention of SFTSV infections in ROK. Our findings demonstrated the development of a high-resolution phylogeographical database of SFTSV for mitigating SFTS outbreaks to humans in the endemic areas.
Genetic reassortment confers a capacity to generate novel variants by which segmented RNA viruses shuffle the viral genomes [52]. Genetic reassortment events promote alterations in viral characteristics including host immunity evasion, transmissibility, and virulence to humans [53,54]. Genome exchanges occur among intra- and inter-lineage of SFTSV in nature, resulting in the emergence of new genotypes [55]. Phylogenetic clustering patterns revealed the six pure genotypes (A-F) and nine reassortments (R1–R9) with different rates of mortality, supporting the genotype-dependent pathogenic potential of SFTSV [9,56]. The distinct distribution of SFTSV genotypes may be correlated with varied mortality rates in China (5.3–16.2%), ROK (23.3%), and Japan (27%) [2,7–9]. The majority of Korean SFTSV belonged to genotype B (69.2%), including three different sub-genotypes; the most dominant sub-lineage was B-2 (36.1%), followed by B-3 (21.1%) and B-1 (12%) [9]. In this study, the phylogenies of SFTSV Hl20-8 from Cheorwon demonstrated incongruent phylogenetic patterns of the tripartite genomes, indicating the differential evolution of each segment. These results suggest that SFTSV Hl20-8 formed a genomic organization compatible with the B-1 sub-genotype (for the L segment) and the B-2 sub-genotype (for the M and S segments). To better understand the pathogenicity and evolutionary complexity, further studies should conduct continuous collection, epidemiological surveys, and risk assessment of various SFTSV genotypes, ROK.
In conclusion, we developed the multiplex PCR-based NGS for SFTSV from tick samples using nanopore sequencing. The entire genomic sequences of SFTSV were newly recovered from ticks (H. longicornis) collected from Pocheon and Cheorwon, supporting the phylogeographical analysis at high resolution for sophisticated prevention of SFTS outbreaks in ROK. Phylogenetic and reassortment analyses demonstrated that SFTSV Hl20-8 from Cheorwon is a reassortant compatible with B-1 and B-2 sub-genotypes. These results provide important insights into the amplicon-based NGS and genetic diversity for the rapid genome-based diagnosis of SFTSV in endemic outbreaks.
Supporting information
S1 Fig. Reverse-transcription polymerase chain reaction (RT-PCR) to validate isolation of severe fever with thrombocytopenia syndrome virus (SFTSV).
RT-PCR was continually performed to confirm isolation of SFTSV (A) Hl19-31-4 and (B) Hl20-8 strains from supernatants and cells from infected Vero E6 cells.
https://doi.org/10.1371/journal.pntd.0010763.s001
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S2 Fig. The plaque assay of severe fever with thrombocytopenia syndrome virus (SFTSV) Hl19-31-4 and Hl20-8 strains.
SFTSV Hl19-31-4 and Hl20-8 strains were quantified on 6-well plates with Vero E6 monolayers. Each well indicates different dilutions from top-left to right: no dilution, dilutions at 1:101, and 1:102, and bottom-left to right: dilutions at 1:103, 1:104, and 1:105, respectively. The first plaques of SFTSV (A) Hl19-31-4 and (B) Hl20-8 were confirmed at 5 days post-inoculation, and the number of infectious particles was 1.2×106 and 2.0×106 PFU/mL, respectively.
https://doi.org/10.1371/journal.pntd.0010763.s002
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S1 Table. Multiplex polymerase chain reaction primer information for severe fever with thrombocytopenia syndrome virus L, M, and S segments.
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S2 Table. Oligonucleotide primers to obtain 3’and 5’ends genomic sequences of L, M and S segments of severe fever with thrombocytopenia syndrome virus using rapid amplification of cDNA ends polymerase chain reaction in this study.
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S3 Table. Summary of mapped reads and average depth of multiplex polymerase chain reaction-based nanopore sequencing of severe fever with thrombocytopenia syndrome virus.
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S4 Table. Mapped reads and average depth of multiplex polymerase chain reaction-based Illumina sequencing of severe fever with thrombocytopenia syndrome virus.
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S5 Table. Accession numbers of genomic sequences of severe fever with thrombocytopenia syndrome virus used in this study.
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References
- 1. Yu XJ, Liang MF, Zhang SY, Liu Y, Li JD, Sun YL, et al. Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med. 2011;364(16):1523–32. Epub 2011/03/18. pmid:21410387; PubMed Central PMCID: PMC3113718.
- 2. Zhan J, Wang Q, Cheng J, Hu B, Li J, Zhan F, et al. Current status of severe fever with thrombocytopenia syndrome in China. Virol Sin. 2017;32(1):51–62. Epub 2017/03/03. pmid:28251515; PubMed Central PMCID: PMC6598917.
- 3. Takahashi T, Maeda K, Suzuki T, Ishido A, Shigeoka T, Tominaga T, et al. The first identification and retrospective study of severe fever with thrombocytopenia syndrome in Japan. 2014;209(6):816–27. pmid:24231186
- 4. Kim KH, Yi J, Kim G, Choi SJ, Jun KI, Kim NH, et al. Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerg Infect Dis. 2013;19(11):1892–4. Epub 2013/11/12. pmid:24206586; PubMed Central PMCID: PMC3837670.
- 5. Tran XC, Yun Y, Van An L, Kim SH, Thao NTP, Man PKC, et al. Endemic Severe Fever with Thrombocytopenia Syndrome, Vietnam. Emerg Infect Dis. 2019;25(5):1029–31. Epub 2019/04/20. pmid:31002059; PubMed Central PMCID: PMC6478219.
- 6. Peng SH, Yang SL, Tang SE, Wang TC, Hsu TC, Su CL, et al. Human Case of Severe Fever with Thrombocytopenia Syndrome Virus Infection, Taiwan, 2019. Emerg Infect Dis. 2020;26(7):1612–4. Epub 2020/06/23. pmid:32568054; PubMed Central PMCID: PMC7323535.
- 7. Park SW, Han MG, Yun SM, Park C, Lee WJ, Ryou J. Severe fever with thrombocytopenia syndrome virus, South Korea, 2013. Emerg Infect Dis. 2014;20(11):1880–2. Epub 2014/10/24. pmid:25341085; PubMed Central PMCID: PMC4214315.
- 8.
Diseases NIoI. Severe fever with thrombocytopenia syndrome (SFTS) in Japan, as of February 2016. National Institute of Infectious Diseases Tokyo, Japan; 2016.
- 9. Yun SM, Park SJ, Kim YI, Park SW, Yu MA, Kwon HI, et al. Genetic and pathogenic diversity of severe fever with thrombocytopenia syndrome virus (SFTSV) in South Korea. JCI Insight. 2020;5(2). Epub 2019/12/27. pmid:31877113; PubMed Central PMCID: PMC7098714.
- 10.
Korea Disease Control and Prevention Agency (KDCA). Infectious disease portal. Available at: http://www.cdc.go.kr/npt/biz/npp/ist/bass/bassDissStatsMain.do. Accessed 24 December 2021.
- 11. Liu S, Chai C, Wang C, Amer S, Lv H, He H, et al. Systematic review of severe fever with thrombocytopenia syndrome: virology, epidemiology, and clinical characteristics. Rev Med Virol. 2014;24(2):90–102. Epub 2013/12/07. pmid:24310908; PubMed Central PMCID: PMC4237196.
- 12. Liu Q, He B, Huang S-Y, Wei F, Zhu X-QJTLID. Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis. 2014;14(8):763–72. pmid:24837566
- 13. Casel MA, Park SJ, Choi YKJE, Medicine M. Severe fever with thrombocytopenia syndrome virus: emerging novel phlebovirus and their control strategy. 2021;53(5):713–22. pmid:33953322
- 14. Park SW, Song BG, Shin EH, Yun SM, Han MG, Park MY, et al. Prevalence of severe fever with thrombocytopenia syndrome virus in Haemaphysalis longicornis ticks in South Korea. Ticks Tick Borne Dis. 2014;5(6):975–7. Epub 2014/08/29. pmid:25164614.
- 15. Jo YS, Kang JG, Chae JB, Cho YK, Shin JH, Jheong WH, et al. Prevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks Collected from National Parks in Korea. Vector Borne Zoonotic Dis. 2019;19(4):284–9. Epub 2018/11/28. pmid:30481146.
- 16. Yun S-M, Lee W-G, Ryou J, Yang S-C, Park S-W, Roh JY, et al. Severe fever with thrombocytopenia syndrome virus in ticks collected from humans, South Korea, 2013. Emerging infectious diseases. 2014;20(8):1358. pmid:25061851
- 17. Zhang YZ, Zhou DJ, Qin XC, Tian JH, Xiong Y, Wang JB, et al. The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China. J Virol. 2012;86(5):2864–8. Epub 2011/12/23. pmid:22190717; PubMed Central PMCID: PMC3302241.
- 18. Zhu L, Yin F, Moming A, Zhang J, Wang B, Gao L, et al. First case of laboratory-confirmed severe fever with thrombocytopenia syndrome disease revealed the risk of SFTSV infection in Xinjiang, China. Emerging microbes & infections. 2019;8(1):1122–5. pmid:31347462
- 19. Liu Y, Li Q, Hu W, Wu J, Wang Y, Mei L, et al. Person-to-person transmission of severe fever with thrombocytopenia syndrome virus. Vector Borne Zoonotic Dis. 2012;12(2):156–60. Epub 2011/10/01. pmid:21955213.
- 20. Kim WY, Choi W, Park SW, Wang EB, Lee WJ, Jee Y, et al. Nosocomial transmission of severe fever with thrombocytopenia syndrome in Korea. Clin Infect Dis. 2015;60(11):1681–3. Epub 2015/02/20. pmid:25694652.
- 21. Radford AD, Chapman D, Dixon L, Chantrey J, Darby AC, Hall N. Application of next-generation sequencing technologies in virology. J Gen Virol. 2012;93(Pt 9):1853–68. Epub 2012/06/01. pmid:22647373; PubMed Central PMCID: PMC3709572.
- 22. Houldcroft CJ, Beale MA, Breuer J. Clinical and biological insights from viral genome sequencing. Nat Rev Microbiol. 2017;15(3):183–92. Epub 2017/01/17. pmid:28090077; PubMed Central PMCID: PMC7097211.
- 23. Cassedy A, Parle-McDermott A, O’Kennedy RJFiMB. Virus detection: A review of the current and emerging molecular and immunological methods. 2021;8.
- 24. Song DH, Kim WK, Gu SH, Lee D, Kim JA, No JS, et al. Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture-Based Isolates. Am J Trop Med Hyg. 2017;96(2):389–94. Epub 2016/11/30. pmid:27895275; PubMed Central PMCID: PMC5303042.
- 25. Xu X, Bei J, Xuan Y, Chen J, Chen D, Barker SC, et al. Full-length genome sequence of segmented RNA virus from ticks was obtained using small RNA sequencing data. 2020;21(1):1–8. pmid:32938401
- 26. Charre C, Ginevra C, Sabatier M, Regue H, Destras G, Brun S, et al. Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation. Virus Evol. 2020;6(2):veaa075. Epub 2020/12/16. pmid:33318859; PubMed Central PMCID: PMC7665770.
- 27. No JS, Kim W-K, Cho S, Lee S-H, Kim J-A, Lee D, et al. Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues. Scientific reports. 2019;9(1):1–9.
- 28. McNaughton AL, Roberts HE, Bonsall D, de Cesare M, Mokaya J, Lumley SF, et al. Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV). Sci Rep. 2019;9(1):7081. Epub 2019/05/10. pmid:31068626; PubMed Central PMCID: PMC6506499.
- 29. Faria NR, da Silva Azevedo RdS, Kraemer MU, Souza R, Cunha MS, Hill SC, et al. Zika virus in the Americas: early epidemiological and genetic findings. Science. 2016;352(6283):345–9. pmid:27013429
- 30. Park K, Lee SH, Kim J, Lee J, Lee GY, Cho S, et al. Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea. Viruses. 2021;13(5). Epub 2021/06/03. pmid:34066592; PubMed Central PMCID: PMC8148566.
- 31. Brinkmann A, Ulm S-L, Uddin S, Förster S, Seifert D, Oehme R, et al. Amplicov: Rapid whole-genome sequencing using multiplex pcr amplification and real-time oxford nanopore minion sequencing enables rapid variant identification of SARS-CoV-2. Frontiers in microbiology. 2021:1703. pmid:34276587
- 32. Chong ST, Kim HC, Lee I-Y, Kollars TM Jr, Sancho AR, Sames WJ, et al. Comparison of dragging and sweeping methods for collecting ticks and determining their seasonal distributions for various habitats, Gyeonggi Province, Republic of Korea. Journal of Medical Entomology. 2013;50(3):611–8. pmid:23802457
- 33.
Yamaguti N, Tipton VJ, Keegan HL, Toshioka S. Ticks of Japan, Korea, and the Ryukyu islands. Brigham Young University Science Bulletin, Biological Series. 1971;15(1):1.
- 34. Saito Y, Hoogstraal H, Wassef HY. The Haemaphysalis ticks (Ixodoidea: Ixodidae) of birds. 4. H. (Ornithophysalis) phasiana sp. n. from Japan. J Parasitol. 1974;60(1):198–208. Epub 1974/02/01. pmid:4814793.
- 35. Perez-Sautu U, Gu SH, Caviness K, Song DH, Kim YJ, Paola ND, et al. A Model for the Production of Regulatory Grade Viral Hemorrhagic Fever Exposure Stocks: From Field Surveillance to Advanced Characterization of SFTSV. Viruses. 2020;12(9). Epub 2020/09/03. pmid:32872451; PubMed Central PMCID: PMC7552075.
- 36. Li Z, Cui L, Zhou M, Qi X, Bao C, Hu J, et al. Development and application of a one-step real-time RT-PCR using a minor-groove-binding probe for the detection of a novel bunyavirus in clinical specimens. Journal of medical virology. 2013;85(2):370–7. pmid:23212930
- 37. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–4. Epub 2016/03/24. pmid:27004904; PubMed Central PMCID: PMC8210823.
- 38. Nagarajan N, Kingsford C. GiRaF: robust, computational identification of influenza reassortments via graph mining. Nucleic Acids Res. 2011;39(6):e34. Epub 2010/12/24. pmid:21177643; PubMed Central PMCID: PMC3064795.
- 39. Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. 2001;17(8):754–5. Epub 2001/08/29. pmid:11524383.
- 40. Grubaugh ND, Ladner JT, Lemey P, Pybus OG, Rambaut A, Holmes EC, et al. Tracking virus outbreaks in the twenty-first century. Nature microbiology. 2019;4(1):10–9. pmid:30546099
- 41. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE, Andino R. Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature. 2006;439(7074):344–8. pmid:16327776
- 42. Gardy JL, Loman NJ. Towards a genomics-informed, real-time, global pathogen surveillance system. Nat Rev Genet. 2018;19(1):9–20. Epub 2017/11/14. pmid:29129921; PubMed Central PMCID: PMC7097748.
- 43. Hoenen T, Groseth A, Rosenke K, Fischer RJ, Hoenen A, Judson SD, et al. Nanopore sequencing as a rapidly deployable Ebola outbreak tool. 2016;22(2):331.
- 44. de Jesus JG, Giovanetti M, Rodrigues Faria N, Alcantara LCJ. Acute Vector-Borne Viral Infection: Zika and MinION Surveillance. Microbiol Spectr. 2019;7(4). Epub 2019/08/11. pmid:31400093.
- 45. Page AJ, Mather AE, Le-Viet T, Meader EJ, Alikhan NF, Kay GL, et al. Large-scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management. Microb Genom. 2021;7(6). Epub 2021/06/30. pmid:34184982; PubMed Central PMCID: PMC8461472.
- 46. Yoo JR, Heo ST, Song SW, Bae SG, Lee S, Choi S, et al. Severe Fever with Thrombocytopenia Syndrome Virus in Ticks and SFTS Incidence in Humans, South Korea. Emerg Infect Dis. 2020;26(9):2292–4. Epub 2020/08/21. pmid:32818414; PubMed Central PMCID: PMC7454071.
- 47. Yun SM, Lee WG, Ryou J, Yang SC, Park SW, Roh JY, et al. Severe fever with thrombocytopenia syndrome virus in ticks collected from humans, South Korea, 2013. Emerg Infect Dis. 2014;20(8):1358–61. Epub 2014/07/26. pmid:25061851; PubMed Central PMCID: PMC4111194.
- 48. Park SW, Ryou J, Choi WY, Han MG, Lee WJ. Epidemiological and Clinical Features of Severe Fever with Thrombocytopenia Syndrome During an Outbreak in South Korea, 2013–2015. Am J Trop Med Hyg. 2016;95(6):1358–61. Epub 2016/12/09. pmid:27928084; PubMed Central PMCID: PMC5154450.
- 49. Seo MG, Noh BE, Lee HS, Kim TK, Song BG, Lee HI. Nationwide Temporal and Geographical Distribution of Tick Populations and Phylogenetic Analysis of Severe Fever with Thrombocytopenia Syndrome Virus in Ticks in Korea, 2020. Microorganisms. 2021;9(8). Epub 2021/08/28. pmid:34442708; PubMed Central PMCID: PMC8399818.
- 50. Kim WK, No JS, Lee D, Jung J, Park H, Yi Y, et al. Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus. Clin Infect Dis. 2020;70(3):464–73. Epub 2019/03/21. pmid:30891596.
- 51. Kim WK, Cho S, Lee SH, No JS, Lee GY, Park K, et al. Genomic Epidemiology and Active Surveillance to Investigate Outbreaks of Hantaviruses. Front Cell Infect Microbiol. 2020;10:532388. Epub 2021/01/26. pmid:33489927; PubMed Central PMCID: PMC7819890.
- 52. McDonald SM, Nelson MI, Turner PE, Patton JT. Reassortment in segmented RNA viruses: mechanisms and outcomes. Nat Rev Microbiol. 2016;14(7):448–60. Epub 2016/05/24. pmid:27211789; PubMed Central PMCID: PMC5119462.
- 53. Vijaykrishna D, Mukerji R, Smith GJ. RNA Virus Reassortment: An Evolutionary Mechanism for Host Jumps and Immune Evasion. PLoS Pathog. 2015;11(7):e1004902. Epub 2015/07/15. pmid:26158697; PubMed Central PMCID: PMC4497687.
- 54. Iturriza-Gómara M, Isherwood B, Desselberger U, Gray JJJov. Reassortment in vivo: driving force for diversity of human rotavirus strains isolated in the United Kingdom between 1995 and 1999. 2001;75(8):3696–705. pmid:11264359
- 55. Ding F, Zhang W, Wang L, Hu W, Soares Magalhaes RJ, Sun H, et al. Epidemiologic features of severe fever with thrombocytopenia syndrome in China, 2011–2012. Clin Infect Dis. 2013;56(11):1682–3. Epub 2013/02/23. pmid:23429379.
- 56. Fu Y, Li S, Zhang Z, Man S, Li X, Zhang W, et al. Phylogeographic analysis of severe fever with thrombocytopenia syndrome virus from Zhoushan Islands, China: implication for transmission across the ocean. Sci Rep. 2016;6:19563. Epub 2016/01/26. pmid:26806841; PubMed Central PMCID: PMC4726339.