Data collection
This study was conducted in three cities (Lianyungang, Zhenjiang and Yancheng) and four counties (Yixing, Lishui, Dongtai and Xuyi ), Jiangsu province, Eastern China, the main SFTS epidemic area where human SFTS cases had been reported before(Fig. 1). We randomly selected 10 villages as study site. The wild animals were selected in every study site, including small wild mammals (rodents and hedgehogs) and avian (pheasants).
A total of 1355 serum of wild animals were collected from 2014 to 2019. Blood samples from wild animals were collected directly in serum tubes. The samples were centrifuged at 2560 × g for two minutes and the serum was transferred to small vials, which were kept at − 18 ◦ C until the time of analysis. Rodents, pheasants and hedgehogs were collected with live traps in accordance with standard protocols, as previously described[16–17]. Trapping grids were set up at sites adjacent to case households and in locations chosen to provide geographic diversity. We abided by established safety guidelines for rodent capture and processing. All trapped animals were anesthetized using ketamine, blood was drawn from the retro-orbital sinus.
SFTSV-RNA extraction and real-time RT-PCR.
Total RNA prepared from the serum samples from patients were extracted using an RNeasy kit (Qiagen, Germany) according to the manufacturer’s instructions. Real-time RT-PCR was performed using the QuantiTech RT-PCR kit (Qiagen, Germany). The primers were designed as previously described and used in a one-step real-time RT-PCR. The primers and MGB probe used in the real-time RT-PCR were targeted to the S segment of the viral genome [18]. Conditions for the reaction were as follows: 50°C for 30 min, 95°C for 15 min, 40 cycles at 95°C for 15 sec, and 55°C for 40 sec. Amplification and detection were performed with an Applied Biosystems 7500 Real-time PCR system (Applied Biosystems, Foster City, CA). Data were analyzed using the software supplied by the manufacturer.
ELISA for SFTSV antibody detection.
Serum samples from patients were tested for SFTSV IgG and IgM antibodies with commercial ELISA kits from DAAN GENE(Zhongshan, China). For initial screening, an 1:40 diluted serum sample was used to determine whether the sample was positive for viral antibodies. Positive serum samples were further diluted in a 2-fold serial dilution starting at 1:80 for the assay to obtain endpoint titers determined by the cutoff values set by positive and negative controls as provided with the ELISA kit.
Statistical analysis
SPSS 25.0 (Chicago, IL, USA) was used for all statistical analysis and statistical significance level was set at 0.05. Pearson chi-square test was conducted to compare the detection rates of SFTSV RNA and total antibodies among samples. The McNemar test was applied to compare SFTSV RNA and total antibodies detection rates in the paired specimens, and the kappa value was calculated to compare the detection consistency of the two methods.