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. 2018 Feb;24(2):249-257.
doi: 10.3201/eid2402.171216.

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats

Won-Keun Kim, Jin Sun No, Seung-Ho Lee, Dong Hyun Song, Daesang Lee, Jeong-Ah Kim, Se Hun Gu, Sunhye Park, Seong Tae Jeong, Heung-Chul Kim, Terry A Klein, Michael R Wiley, Gustavo Palacios, Jin-Won Song

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats

Won-Keun Kim et al. Emerg Infect Dis. 2018 Feb.

Abstract

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR-based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000-2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Keywords: RT-PCR; Seoul virus; South Korea; global diversity; hantavirus; humans; multiplex PCR; next-generation sequencing; orthohantavirus; phylogenetic analysis; rats; real-time quantitative PCR; reverse transcription PCR; viruses.

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Figures

Figure 1
Figure 1
Measurement of SEOV RNA loads in different tissues of Rattus norvegicus rats, South Korea, 2000–2016. Ct values were determined for SEOV small segment RNA in lung, liver, kidney, and spleen tissues obtained from 5 rats positive for SEOV IgG and SEOV RNA. Solid horizontal line indicates assay cutoff value. Ct, cycle threshold; S, small; SEOV, Seoul virus.
Figure 2
Figure 2
Phylogenetic analysis of SEOV small RNA segments, South Korea, 2000–2016, and reference strains. A phylogenetic tree was generated by using the maximum-likelihood method with the T92 + gamma distribution model of evolution and alignment of small RNA segment sequences (nt 193–1332) of SEOV strains. Colored groups indicate the areas where SEOV strains were identified: group A, northeastern and southeastern China and North Korea; group B, Europe (France and Belgium) and Southeast Asia (Vietnam and Singapore); group C, South Korea, Japan, and the United States; group D, southeastern China; group E, United Kingdom and the United States; group F, mountainous areas in southeastern China. Bold red indicates SEOV strains sequenced in this study. Topologies were evaluated by bootstrap analyses of 1,000 iterations. Numbers along branches are bootstrap values. GenBank accession numbers are provided. Scale bar indicates nucleotide substitutions per site. SEOV, Seoul virus.
Figure 3
Figure 3
Phylogenetic analysis of SEOV medium RNA segments, South Korea, 2000–2016, and reference strains. A phylogenetic tree was generated by using the maximum-likelihood method with the general time reversible + gamma + invariant model of evolution and alignment of medium segment sequences (nt 47–3430) of SEOV strains. Colored groups indicate the areas where SEOV strains were identified: group A, northeastern and southeastern China and North Korea; group B, Europe (France and Belgium) and Southeast Asia (Vietnam and Singapore); group C, South Korea, Japan, and the United States; group D, southeastern China; group E, United Kingdom and the United States; group F, mountainous areas in southeastern China. Bold red indicates SEOV strains sequenced in this study. Topologies were evaluated by bootstrap analyses of 1,000 iterations. Numbers along branches are bootstrap values. GenBank accession numbers are provided. Scale bar indicates nucleotide substitutions per site. SEOV, Seoul virus.
Figure 4
Figure 4
Phylogenetic analysis of SEOV large RNA segments, South Korea, 2000–2016, and reference strains. A phylogenetic tree was generated by using the maximum-likelihood method with the TN93 + gamma + invariant model of evolution and alignment of large segment sequences (nt 1–6510) of SEOV strains. Colored groups indicate the areas where SEOV strains were identified: group A, southeastern China and North Korea; group B, Europe (France); group C, South Korea and the United States; group E, United Kingdom and the United States. Bold red indicates SEOV strains sequenced in this study. Topologies were evaluated by bootstrap analyses of 1,000 iterations. Numbers along branches are bootstrap values. GenBank accession number are provided. Scale bar indicates nucleotide substitutions per site. SEOV, Seoul virus.
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