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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.

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