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HTNV outbreaks are particularly prevalent in the Gyeonggi and Gangwon Provinces of the Republic of Korea (ROK). This study aimed to evaluate the application of advanced nanopore sequencing and bioinformatics to generate complete genome sequences of HTNV, with the objective of accurately identifying infection sources and analyzing their genetic diversity.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methodology\/Principal findings<\/jats:title>\n<jats:p>In 2022 and 2023, we collected 579 small mammals from 11 distinct locations across Gyeonggi and Gangwon Provinces, as well as in Gwangju Metropolitan City, ROK. Among these, 498 <jats:italic>Apodemus agrarius<\/jats:italic> specimens were subjected to an epidemiological survey to investigate HTNV infections. The serological and molecular positivity of HTNV were found to be 65\/498 (13.1%) and 17\/65 (26.2%), respectively. Furthermore, 15 whole-genome sequences of HTNV were obtained from rodents in Gyeonggi and Gangwon Provinces. We developed a novel amplicon-based nanopore sequencing approach to acquire high-fidelity and precise genomic sequences of HTNV. Genome exchange analysis revealed three reassortant candidates, including heterogeneous L segments, from Paju-si and Yeoncheon-gun in Gyeonggi Province.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Conclusion\/Significance<\/jats:title>\n<jats:p>Our findings enhance the resolution of the spatiotemporal genomic surveillance of HTNV by consistently providing new viral sequences and epidemiological data from HFRS-endemic regions in the ROK. This report signifies a notable advancement in nanopore sequencing techniques and bioinformatics, offering a robust platform for genome-based diagnostics and sophisticated phylogenetic analyses of orthohantaviruses, which are essential for public health strategies aimed at controlling HFRS.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0012859","type":"journal-article","created":{"date-parts":[[2025,2,7]],"date-time":"2025-02-07T18:40:17Z","timestamp":1738953617000},"page":"e0012859","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":8,"title":["Epidemiological surveillance and phylogenetic diversity of Orthohantavirus hantanense using high-fidelity nanopore sequencing, Republic of 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