A retrospective survey of the seroprevalence of severe fever with thrombocytopenia syndrome virus in wild animals in Japan

Abstract Severe fever with thrombocytopenia syndrome (SFTS) has a high fatality rate and is caused by SFTS virus (SFTSV). Currently, SFTS is endemic to some areas in western Japan, and wild animals are considered to play important roles in the circulation of SFTSV in the environment. Previous retrospective surveys using samples mainly obtained between 2006 and 2015 revealed serological evidence of SFTSV infection in wild animals; however, seroprevalence before 2006 remains unclear. In this study, we investigated the presence of anti‐SFTSV antibodies in a total of 521 serum samples from nine wild animal species collected from 11 prefectures in central and eastern Japan between 1980 and 2000. All samples yielded negative results for antibodies to SFTSV, suggesting that there had been few or no SFTSV infections before 2000 in the sampled areas.


| MATERIAL S AND ME THODS
A total of 521 serum samples were collected from nine wild animal species in 11 prefectures throughout the largest island of Japan, Honshu, which were used in previous studies ( Inoshima et al., 1999( Inoshima et al., , 2001( Inoshima et al., , 2002Suzuki et al., 1993) (Table 1 and Table S1, and Figure 1).
Titres of antibodies to SFTSV antigen were determined using an enzyme-linked immunosorbent assay (ELISA) using protein A/G conjugated with horseradish peroxidase. Briefly, half of the wells of the ELISA plates (Nunc, Roskilde, Denmark) were coated with a predetermined optimal quantity (approximately 100 ng/well) of lysates of SFTSV-infected Huh7 cells, and the remaining wells were coated with negative control antigens (lysates of Huh7 mock-infected cells). The antigens used in the ELISAs were prepared as described previously (Kimura et al., 2018;Park et al., 2019). OD value exceeded the cut-off value (mean + 3SD). Negative control sera of normal rabbits, rats and mice typically showed OD values between −0.14 and 0.13 in this assay.

| RE SULTS AND D ISCUSS I ON
In every experiment, reference control sera showed high OD values for SFTSV-antigen at a dilution of 1 in 100 and 400. In every experiment, the signal/background ratios were above 5.5. After subtraction of the standardized background OD values, the reference sample remained at OD values between 1.37 and 1.85 in all experiments. However, all of the 521 samples (Table 1), showed low reactivity, and their background subtraction was below the cut-off value (0.33). The presence of antibodies to SFTSV in wild animals was considered as evidence of past SFTSV infection. Thus, these results indicated that SFTSV infection was non-existent or rare before 2000, in these animal species in the tested areas in Japan.
In the present study, SFTSV antibodies were not detected in SFTSV antibodies were also detected in Japanese wild boars in Nagasaki Prefecture, whose blood samples were collected be- In conclusion, in the present retrospective study, we did not find SFTSV infections in any of the nine wild animal species examined between 1980 and 2000 in central and eastern Japan. Thus, the humans who handled the wild animals in previous studies (Inoshima et al., 1999(Inoshima et al., , 2001(Inoshima et al., , 2002Suzuki et al., 1993) AMED (18fk0108002, 18fk0108017, 18fk0108069, and 19fk0108081).

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest.

E TH I C A L S TATEM ENT
Sample collection was carried out according to the animal welfare code of Japan.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.400.