Ethics statement
Small mammal trapping and handling procedures were conducted in full compliance with relevant institutional and national guidelines and regulations. Specifically, the experimental protocols involving animals were reviewed and approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC) under the following protocol numbers: #2010–212, #2016–49, and #2019–4. Field collection of wild rodents was carried out by Force Health Protection and Preventive Medicine, 65th Medical Brigade, 8th US Army, US Forces Korea (USFK), in accordance with USFK Regulation 40–1, which governs the “Prevention, Surveillance, and Treatment of Hemorrhagic Fever with Renal Syndrome” at US military installations and US/ROK-operated military training sites. Standardized protocols were followed to minimize risks associated with potentially infected animals. All laboratory personnel handling rodents were vaccinated with the Korean-authorized Hantavax vaccine. Small mammals were euthanized via cardiac puncture under isoflurane anesthesia in accordance with approved IACUC protocols. All procedures adhered to the ARRIVE guidelines (https://arriveguidelines.org) and complied with all applicable ethical standards for animal research. Every effort was made to minimize animal suffering throughout the study.
Habitat description
Small mammal surveillance was conducted at 32 sites, including US military installations/air bases, TAs, LFCs, and a communications post from November 2000 through December 2019 (Supplementary Table 4). Additionally, a civilian dairy farm in Singye-ri, Haenam-si, Jeollanam Province, was surveyed in this study. The topography and ecology of the training sites varied, influenced in part by military activities and designations. These areas ranged from flat, low-lying regions to mountainous, rocky multipurpose range complexes, as well as narrow and expanded river valleys with unmanaged grasses and forested hillsides. Over the survey period, multipurpose TAs/LFCs were modernized and characterized by hardened, heavily graveled roads and short-cut grasses, with limited unmanaged tall grasses, herbaceous and scrub vegetation, or forested hillsides. Some areas, such as the Dagmar North TA in Jangjwa-ri, consisted entirely of unmanaged lands. Military operations at these sites included command and control exercises, weapons firing, and limited maneuvers at smaller training sites. Larger multipurpose TAs and LFCs hosted personnel and mechanized infantry maneuvers, artillery and rocket impact zones, and air assault, helicopter, and aircraft training activities. Agricultural activities surrounding the TAs included land cultivation beginning in March, irrigation, planting, and harvesting of rice, fruit, and vegetable crops, as well as tree removal and occasional burning and cutting of tall grasses and trees along the military perimeters. Civilian encroachment onto training sites also occurred but has been limited as perimeter fences have been installed at many areas.
Sampling
Sherman live capture folding traps (7.5 × 8.75 × 22.5 cm; H.B. Sherman, Tallahassee, FL, USA), baited with peanut butter placed between two saltine crackers, were set during daylight hours (12:00–17:00) and collected the following morning as previously described39. Global positioning system coordinates and elevations were recorded at both the beginning and end of each trap line (20–60 traps set at 4–5 m intervals). Field collection records, including photographs of the collection site, were entered into an electronic database (Microsoft Excel, Microsoft Corp., Redmond, WA, USA) to document each trap line along with vegetation density, diversity, and composition at the time of the survey. Traps positive for small mammals were numbered sequentially and placed in secured containers for transport to an ABSL-3 laboratory, Korea University, Seoul, ROK. In the laboratory, each small mammal was assigned a unique identification number, euthanized, identified to species, sexed, weighed, and examined for gravid status (2002–2019). Spleen, lung, and kidney samples were placed in separate cryovials, and all blood and tissue samples were stored at −70 °C until processed for diagnostic testing.
Serological screening
Indirect immunofluorescence Ab testing was performed to detect IgG Abs against HTNV in rodent sera. Briefly, Vero E6 cells (ATCC, DR-L2785) infected with HTNV were fixed on multi-well slides using cold acetone for 10 min. A total of 25 µL of 1:32 diluted rodent sera in phosphate-buffered saline (PBS) was added to each well, followed by incubation at 37 °C for 30 min. The antigen slides were washed twice with PBS, and 25 µL of fluorescein isothiocyanate-conjugated anti-mouse IgG (ICN Pharmaceuticals, Laval, Canada) was added to each well. After incubation and washing, the slides were mounted with glycine-buffered glycerol under cover slips and examined under a fluorescence microscope (Axio Scope; Zeiss, Berlin, Germany).
Statistical analysis
Chi-square (χ2) analyses were performed using GraphPad Prism 10.4.1 for Windows (GraphPad Software, Boston, MA, USA; www.graphpad.com).
Risk assessment for hantavirus infection
To evaluate HFRS risk across multiple rodent survey sites, several parameters were considered, including HTNV prevalence in rodent populations, disease severity, environmental factors (e.g., dirt and gravel roads, unmanaged lands), rodent bionomics (capture rates, peak reproductive periods, distribution, and habitat characteristics), HTNV Ab-seropositivity rates, rodent density (estimated number of HTNV Ab-positive rodents per 100 traps), transmission dynamics, potential for human exposure, human activities, and available protective measures. Risk levels were classified as high, moderate, or low.
