We performed a cross-sectional seroepidemiological study of SFTSV among healthy farmers from various rural areas of Jeju Island over a period of three years from January 2015 to December 2017 (Fig. 1). Jeju Island (33°0′ N, 126°0′ E) is the largest island located off the coast of the Korean Peninsula. It has a humid subtropical climate and is warmer than the rest of Korea (daily mean temperature: 15.8–16.6°C). The climate in winter is moderate with temperatures rarely falling below 0°C (32°F). A total of 650,000 people reside on the island, about 15% of whom are farmers.

The study subjects were farmers living on the Jeju Island who were some of the participants of the Prevention for Agricultural Injury of Farmers study which was conducted by the Center for Farmers' Safety and Health at Jeju National University Hospital. Along with the team of the Prevention for Agricultural Injury of Farmers study, we recruited participants and sequentially tested them from 2015 to 2017, using a three-step protocol. In the first step, ten rural villages were selected based on the type of agriculture and distance from urban areas; a total of 400 farmers (50 from each village) were selected. In the second step, 377 of these 400 farmers verbally consented to participate via telephone. In the third step, trained researchers visited each village and conducted face-to-face interviews among the participants. Three hundred and twenty-four farmers were enrolled in the cohort and 53 were excluded because they refused to participate in the face-to-face interviews. Finally, 254 farmers agreed to provide blood samples for SFTSV antibody tests and were included in the study. All subjects provided written informed consent. The study protocol was approved by the Institutional Review Board of Jeju National University Hospital (IRB no. 2015-11-002-004).

Data regarding sociodemographic status, medical history, occupation, type of farming, history of tick bites, history of SFTS, history of contact with SFTS patients, and other relevant information were collected via standardized questionnaires and interviews. The team analyzed blood samples collected from subjects for the presence of SFTSV antibodies.

Serum samples obtained from the subjects were stored until serologic analyses could be performed. SFTSV IgG was detected using an enzyme-linked immunosorbent assay (ELISA), as previously described [9]. In our previous study, the sensitivity and specificity of ELISA were comparable to those of the immunofluorescence assay (IFA); samples that tested positive for the IgG in the ELISA also showed positive results in the IFA. Huh7 cells were infected with SFTSV strain HB 29 (multiplicity of infection = 0.1) and incubated at 37°C for 48 h. The cells were collected and washed with phosphate-buffered saline (PBS) and lysed with PBS containing 1% NP-40. The cell lysates were centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected and used as a source of SFTSV antigens for the IgG ELISA. Huh7 cell lysates of uninfected cells were used as a negative control. A Nunc-Immuno Plate (Thermo Fisher Scientific, Waltham, MA, USA) was coated with a predetermined optimal quantity of cell lysates from SFTSV-infected or uninfected Huh7 cells (1:800 dilution with PBS) and incubated overnight at 4°C. The cut-off value was set as the average value obtained in the control sera (serum samples obtained from healthy donors) plus thrice the standard deviation (SD) (mean ± 3 × SD). An absorbance reader (Sunrise, Tecan's Magellan^TM, Männedorf, Switzerland) was used to measure the absorbance in the ELISA. A sample was considered antibody-positive if it yielded an optical density (OD) above the predetermined cut-off value at 405 nm (OD₄₀₅). The average OD values for the negative and positive controls were ~0.12 and ~1.06, respectively. The antibody-positive serum of an SFTS patient who was admitted at our hospital was used as a positive control (OD₄₀₅ = 1.06). The SFTSV IgG antibody levels in the specimens obtained from the cohort were higher than that in the positive control (OD₄₀₅ = 1.06).

Statistical analyses were performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). The results for categorical variables are presented as frequencies or proportions and were compared using the chi-square test. P <0.05 was considered statistically significant. The results for continuous variables are presented as the means ± standard deviations or medians and ranges. Regional annual incidences were calculated using the annual number of cases of SFTS reported in the Disease Web Statistics System, Korea Centers for Disease Control and Prevention (http://is.cdc.go.kr) and population statistics based on resident registration (http://www.kosis.kr).

A total of 254 healthy subjects were enrolled in the study, and 421 samples were collected. Figure 1 shows the geographic distribution of all the healthy subjects. Many of the subjects had resided in the same location for at least 10 years. Five samples were obtained in Jeju-si, 67 in Jocheon-eup, 139 in Aewol-eup, 98 in Nanwon-eup, 39 in Hangyeong-myeon, 44 in Seongsan-eup, 2 in Daejeong-eup, 10 in Hanlim-eup, 2 in Gujwa-eup, 1 in Pyoseon-myeon, and 12 in Gangjeong-dong. All samples were subjected to ELISA for SFTSV detection. We obtained 131 samples in 2015, 167 in 2016, and 123 in 2017. Tests were performed once for 142 subjects, twice for 83 subjects, and thrice for 72 subjects from 2015 to 2017. The mean age of the participants was 59.9 years (range, 27–84 years; 68.9% male). Thirteen participants farmed grain, 120 farmed vegetables, 125 farmed fruits, 27 farmed livestock, and 8 engaged in other forms of farming. Only eight samples tested positive for SFTSV IgG, resulting in an overall prevalence rate of 2.4% (6/254 subjects). One subject showed positive results over the entire three year period.

Six patients and eight samples tested positive for SFTS IgG (Table 1), although all seropositive samples tested negative for the S segment of SFTSV in a reverse transcriptase polymerase chain reaction assay. The areas with the highest SFTSV IgG seropositivity rates were Seonheul-ri in Jocheon-eup, followed by Namwon-eup, which are also the regions in which SFTS was most common and had the highest number of reported SFTS-related deaths in 2013 and 2015. Fruit farmers were at a higher risk of exposure to SFTSV than were other farmers such as those who farmed grain, vegetable, and livestock, and those who engaged in other forms of farming (4.8% vs 0%; P <0.001). Farmers in Jocheon-eup also had a significantly higher exposure risk than those in other locations. The subjects who tested positive for SFTSV IgG were fruit farmers who cultivated mandarins. There was no significant difference in the proportion of participants who wore protective equipment between the seropositive and seronegative groups. Additionally, fruit farmers with vinyl greenhouses tended to have a lower seropositivity rate of SFTS IgG (P = 0.09). There were no significant differences in the other characteristics between the seropositive and seronegative groups. The subjects had no history of tick bites or contact with SFTS patients. The seropositive group included 4 men and 2 women, none of whom showed typical symptoms or other infectious diseases during the period of sample collection. The median age was 69 (range, 56 – 78) years.