Multiplex PCR-Based Nanopore Sequencing and Epidemiological Surveillance of Hantaan orthohantavirus in Apodemus agrarius, Republic of Korea

Abstract

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

Keywords: Hantaan virus (HTNV); MinION sequencing; amplicon-based NGS; genetic diversity; hemorrhagic fever with renal syndrome (HFRS); next-generation sequencing (NGS); phylogenetic analysis; viral genome surveillance; whole-genome sequencing.

Figure 1

Workflow overview of the amplicon-based nanopore sequencing for Hantaan virus (HTNV) used in this study. Total RNA was extracted from rodent lung tissues, and cDNA was synthesized from 1 µg of total RNA with a random hexamer and OSM55. cDNA was amplified using HTNV-specific primer mixtures for library preparation. Barcoded libraries were pooled, ligated to sequencing adapter, and sequenced using the MinION device with FLO-MIN106 (R9.4) flow cell for 8 h. The basecalled sequences were combined into a single FASTA file, and the reads were mapped to the reference genome sequences. The consensus sequences were extracted for downstream analysis.

Figure 2

Geographical location of the trapping sites of Hantaan virus (HTNV) collected from Gyeonggi and Gangwon Provinces, the Republic of Korea. The geographic map shows different trapping areas where small mammals were captured at Gyeonggi and Gangwon Provinces in 2019. The colored circles indicate the HTNV RNA positive sites: Pocheon, blue (Jangam-ri); Yeoncheon, green (Dosin-ri) and brown (Sang-ri); Cheorwon, orange (Gwanu-ri) and red (Dochang-ri); Hwacheon-gun, violet (Pungsan-ri). The black circles represent the locations where no HTNV RNA was detected: Paju (Jeogam-ri); Pocheon (Jail-ri); Yeoncheon (Hyunga-ri, Dongi-ri, Ohkye-ri, Yangwon-ri, and Bugok-ri); Cheorwon (Eupnae-ri, Munhye-ri, and Chungyang-ri); and Chuncheon (Gamjeong-ri, Sinchon-ri, Jinae-ri, Balsan-ri, and Yulmun-ri). A Quantum Geographical Information System (QGIS) 3.10 for Mac was used to create the map, which was modified in Adobe Illustrator CC 2019.

Figure 3

Average depth of coverage for Hantaan virus (HTNV) obtained from MinION sequencing at different cumulated running times. The illustration shows the average depth of coverage generated from amplicon-based MinION sequencing based on (A) HTNV L segment, (B) M segment, and (C) S segment genomes by using different cumulated running times (2, 4, 8, and 16 h).

Figure 4

Phylogenetic analysis of Hantaan virus (HTNV) whole-genome sequences by using amplicon next-generation sequencing (NGS) and MiSeq and MinION platforms. Phylogenetic trees were generated using maximum-likelihood method and bootstrap of 1000 iterations based on (A) HTNV L segment (1–6533 nt), (B) HTNV M segment (1–3616 nt), and (C) HTNV S segment (1–1696 nt). The numbers at each node are bootstrap probabilities, as determined for 1000 iterations. The newly obtained HTNV are shown in bold lettering and designated by color indicating the specific sites: blue, Jangam-ri of Pocheon; green, Dosin-ri of Yeoncheon; brown, Sang-ri of Yeoncheon; orange, Gwanu-ri of Cheorwon; red, Dochang-ri of Cheorwon; and violet, Pungsan-ri of Hwacheon. The newly acquired HTNV genomic sequences in this study are described in Table S1. The following other HTNV genome sequences were used in the analysis: HTNV Aa03-387 (L segment, KT934958; M segment, KT934992; S segment, KT935026), Aa05-331 (L segment, KT934962; M segment, KT934996; S segment, KT935030), Aa09-410 (L segment, KU207177; M segment, KU207185; S segment, KU207193), Aa09-948 (L segment, KT934966; M segment, KT935000; S segment, KT935034), Aa10-434 (L segment, KT934970; M segment, KT935004; S segment, KT935038), Aa10-518 (L segment, KT934971; M segment, KT935005; S segment, KT935039), Aa14-266 (L segment, KT934979; M segment, KT935013; S segment, KT935047), Aa14-272 (L segment, KT934980; M segment, KT935014; S segment, KT935048), Aa14-362 (L segment, KT934981; M segment, KT935015; S segment, KT935049), Aa14-368 (L segment, KT934982; M segment, KT935016; S segment, KT935050), Aa15-56 (L segment, KU207179; M segment, KU207187; S segment, KU207195), Aa15-58 (L segment, KU207180; M segment, KU207188; S segment, KU207196), Aa15-82 (L segment, MT012572; M segment, MT012560; S segment, MT012548), Aa15-84 (L segment, MT012573; M segment, MT012561; S segment, MT012549), Aa17-353 (L segment, MT012575; M segment, MT012563; S segment, MT012551), Aa17-367 (L segment, MT012576; M segment, MT012564; S segment, MT012552), Aa17-421 (L segment, MT012577; M segment, MT012565; S segment, MT012553), Aa17-422 (L segment, MT012578; M segment, MT012566; S segment, MT012554), Aa18-164 (L segment, MT012579; M segment, MT012567; S segment, MT012555), Aa18-179 (L segment, MT012580; M segment, MT012568; S segment, MT012556), HTNV 76-118 (L segment, NC_005222; M segment, M14627; S segment, M14626), HV004 (L segment, JQ083393; M segment, JQ083394; S segment, JQ093395).