Dab substrate

The YG-1 strain of SFTSV was kindly provided by Ken Maeda, Yamaguchi University. The NagH2013-1 strain of SFTSV was isolated from an SFTS patient in Nagasaki in 2013. Vero E6 cells were maintained in Eagle’s minimal essential medium (EMEM; Nissui Pharmaceutical Co.) containing 10 % fetal bovine serum (FBS). Stock SFTSV was prepared from the cell culture medium of Vero E6 cells in EMEM containing 2 % FBS. Virus titers were determined by a focus forming assay [12 (link)]. Briefly, confluent Vero E6 cells were inoculated with serially diluted culture supernatants of SFTSV and incubated in 2 % FBS EMEM containing 1 % methyl cellulose 4000 (Wako Pure Chemical Industries, Ltd.) for 5 days. Viral foci were detected by using SFTSV antiserum (source: recovered SFTS human case), peroxidase-conjugated antihuman IgG (American Qualex), and the DAB substrate (Wako Pure Chemical Industries, Ltd.). Virus titers were expressed as focus-forming units (ffu) per milliliter. The experiment using human serum was performed with the approval of the ethics committee of the Institute of Tropical Medicine, Nagasaki University (approval number: 140829129). All experiments using live SFTSV were performed in a biosafety level 3 laboratory at Nagasaki University according to standard BSL3 guidelines.

The neutralizing activity of antibodies from the IgG- and IgM-positive sera was confirmed with a 50% focus reduction neutralization test (FRNT₅₀) as described in previous studies [21 (link), 40 (link)]. The heat-treated serum samples were mixed with equal volumes of 40 focus-forming units. After incubation at 37°C for 1 hour, the mixture was transferred to duplicate 96-well plates of confluent Vero cell monolayers. After incubation at 37°C for 1.5 hours, the cells were overlaid with 150 μL of 2% FCS MEM containing 1% methylcellulose 4000 (WAKO Pure Chemical Industries, Japan). The plates were then incubated at 37°C with 5% CO₂ for 36 hours. After fixing the cells, they were blocked and permeabilized as described in previous studies [21 (link)]. Viral foci were detected by immunostaining the cells with anti-CHIKV serum from C57BL/6J mice, peroxidase-conjugated anti-mouse IgG (American Qualex, USA), and DAB substrate (WAKO Pure Chemical Industries, Japan). The endpoint serum dilution that provided a ≥ 50% reduction compared with the mean number of the control well was considered the FRNT₅₀ titer. Confirmed CHIKV cases were defined as IgG- or IgM-positive with a neutralization titer of ≥ 10.