Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture–Based Isolates

Dong Hyun Song The 5th R&D Institute, Agency for Defense Development, Daejeon, Republic of Korea.

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Won-Keun Kim Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea.

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Se Hun Gu The 5th R&D Institute, Agency for Defense Development, Daejeon, Republic of Korea.

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Daesang Lee The 5th R&D Institute, Agency for Defense Development, Daejeon, Republic of Korea.

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Jeong-Ah Kim Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea.

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Jin Sun No Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea.

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Seung-Ho Lee Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea.

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Michael R. Wiley The Center for Genome Science, U.S. Army Medical Research Institute of Infectious Disease, Fort Detrick, Maryland.

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Gustavo Palacios The Center for Genome Science, U.S. Army Medical Research Institute of Infectious Disease, Fort Detrick, Maryland.

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Jin-Won Song Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea.

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Seong Tae Jeong The 5th R&D Institute, Agency for Defense Development, Daejeon, Republic of Korea.

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Hantaan virus (HTNV), identified in the striped field mouse (Apodemus agrarius), belongs to the genus Hantavirus of the family Bunyaviridae and contains tripartite RNA genomes, small (S), medium (M), and large (L) segments. HTNV is a major causative for hemorrhagic fever with renal syndrome (HFRS) with fatality rates ranging from 1% to 15% in the Republic of Korea (ROK) and China. Defining of HTNV whole-genome sequences and isolation of the infectious particle play a critical role in the characterization and preventive and therapeutic strategies of hantavirus outbreaks. Next-generation sequencing (NGS) provides an advanced tool for massive genomic sequencing of viruses. However, the isolation of viral infectious particles is a huge obstacle to investigate and develop anti-virals for hantaviruses. Here, we report 12 HTNV isolates from lung tissues of the striped field mouse in the highly HFRS-endemic areas. Sequence-independent, single-primer amplification (SISPA) NGS was attempted to recover the genomic sequences of HTNV isolates. The nucleotide sequence of HTNV S, M, and L segments were covered up to 99.4–100%, 97.5–100%, and 95.6–99.8%, respectively, based on the full length of the prototype HTNV 76-118. The whole-genome sequencing of HTNV isolates was accomplished by additional reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) PCR. In conclusion, this study will lead to the attempt and usage of SISPA NGS technologies to delineate the whole-genome sequence of hantaviruses, providing a new era of viral genomics for the surveillance, trace, and disease risk management of HFRS incidents.

Author Notes

* Address correspondence to Jin-Won Song, Department of Microbiology, College of Medicine, Korea University, Inchon-ro 73, Seongbuk-gu, Seoul, Republic of Korea 02841, E-mail: jwsong@korea.ac.kr or Seong Tae Jeong, The 5th R&D Institute, Agency for Defense Development, Yuseong, P.O. Box 35, Daejeon, Republic of Korea 34188, E-mail: seongtae@add.re.kr.
† These authors equally contributed to this study.

Financial support: This study was funded by Agency for Defense Development (UE134020ID/UD160022ID).

Authors' addresses: Dong Hyun Song, Se Hun Gu, Daesang Lee, and Seong Tae Jeong, The 5th R&D Institute, Agency for Defense Development, Daejeon, Republic of Korea, E-mails: swpia@add.re.kr, sehungu@add.re.kr, dslee@add.re.kr, and seongtae@add.re.kr. Won-Keun Kim, Jeong-Ah Kim, Jin Sun No, Seung-Ho Lee, and Jin-Won Song, Department of Microbiology, College of Medicine, Korea University, Seoul, Republic of Korea, E-mails: wkkim1061@korea.ac.kr, youminlove3@korea.ac.kr, dybono@korea.ac.kr, leeds1104@korea.ac.kr, and jwsong@korea.ac.kr. Michael R. Wiley and Gustavo Palacios, The Center for Genome Science, U.S. Army Medical Research Institute of Infectious Disease, Fort Detrick, MD, E-mails: michael.r.wiley19.ctr@mail.mil and gustavo.f.palacios.ctr@mail.mil.

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