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We collected sera from 215 deer (5 to 10 samples per farm) on farms throughout South Korea from March of 2015 to October of 2017 (Fig. 1). To detect the M gene segment of SFTSV, we conducted RT-PCR with total RNA extracted from each sample. Two sera samples were positive for viral RNA, suggesting active infections of Korean deer during this time. To isolate the virus, we attempted to infect Vero E6 cells with the positive sera; however, no virus was isolated from the cell cultures following three blind passages. The M gene sequencing results revealed that the two viruses belonged to the B genotype and they showed the highest homology with KADGH/Human/Korea strain (97.0% nucleotide identity), which was isolated from a human serum sample in South Korea in 2013 (Fig. 2).
Figure 1. Sampling locations of domesticated deer serum in South Korea. Deer sera were collected from farms in Chungnam (A), Chungbuk (B), Gyeongbuk (C) and Jeonbuk (D) provinces marked by blue in Korea respectively.
Figure 2. Phylogenetic analysis based on the nucleotide sequences of M segment of SFTSV strains. This figure shows the phylogenetic relationship of the M gene segment within SFTSV. The numbers on the branches indicate bootstrap percentages based on 1000 replications. The scale bar indicates the nucleotide substitutions per site and the tree was generated with the Maximum Likelihood (ML) method based on the Kimura 2-parameter model using MEGA Version 7.0 software. The branches were designated as SFTSV strains belonging to the genotypes A, B, C, D, E, and F, respectively. The deer-isolated SFTSV sequences analyzed in this study are marked with red closed circles. Sequenced data have been deposited in GenBank under the following accession numbers: Y16 (MK955794) and K12 (MK955793). These sequence will be released when the print version release in public.
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To evaluate the sero-prevalence of SFTSV in domesticated deer in South Korea, deer sera were evaluated for the presence of the SFTSV antibody using two different ELISAs, rN-and Gn-based. With the rN-based ELISA, 30 (14.0%) of the 215 samples were found to be positive (O.D value greater than 0.7). The rGn-based ELISA showed 7.9% (17 of 215 specimens) of samples were positive (O.D greater than 0.7) for this surface protein. It should be noted that while only one serum sample was positive for SFTSV Gn antibody in 2015, the SFTSV sero-positive rates increased to 12.1% in 2016 and 28.2% in 2017 (Table 1). Moreover, the two samples found to be positive by RTPCR only exhibited a positive result in the rGn-based ELISA, suggesting increased genotype-specific sensitivity of the rGn-based ELISA. Overall, 40 samples were found to be positive by ELISA (Table 1). These data suggest that about 18.6% of domesticated deer were exposed to SFTSV infection over the three years surveyed and thus, exhibit potential as a reservoir for SFTSV during zoonotic infection.
Years Positive Negative (both ELISAs) Total rN* positive rGn+ positive rN and rGn Positive rN or rGn Positive 2015 0 (0.0%) 1 (2.6%) 0 (0.0%) 1 (2.6%) 38 (97.4%) 39 (100%) 2016 6 (9.1%) 3 (4.5%) 1 (1.5%) 8 (12.1%) 58 (87.9%)# 66 (100%) 2017 24 (21.8%)# 13 (11.8%) 6 (5.5%) 31 (28.2%) 79 (71.8%)## 110 (100%) Total 30 (14.0%) 17 (7.9%) 7 (3.3%) 40 (18.6%) 175 (81.4%) 215 (100%) *rN, recombinant N protein-based ELISA; +rGn, recombinant Gn protein-based ELISA.
#Indicates statistically significant differences in positive or negative rates compared with 2015 isolates as determined by Gehan–Breslow– Wilcoxon test. Statistical analyses were performed using GraphPad Prism version 8.00 for Windows (GraphPad Software, La Jolla, CA) (#indicates P < 0.05, ##indicates P < 0.0001).Table 1. The result of rN and Gn protein-based ELISAs of domestic deer serum samples.
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To confirm the results of the ELISAs, IFA was performed with the 40 SFTSV-positive deer sera. Vero E6 cells in 6-well plates were infected with 1 × 103 TCID50/mL CB1 SFTSV strains (genotype B) and primary deer sera (1:20 dilutions) were incubated with cells for 3 h. Following the incubation, fluorescence was detected using fluoresceinconjugated secondary deer antibodies and fluorescence microscopy. For positive and negative controls, an in-house generated ferret antisera was used (described elsewhere) (Yu et al. 2018b). The IFA results show that all 30 rNand 17 rGn-based ELISA positive sera were clearly IFA positive (Fig. 3).
Figure 3. Immunofluorescence assay (IFA) of deer serum. IFA was performed using the rNor rGn-based ELISA positive or negative deer serum. Vero E6 cells were infected with 1 × 103 TCID50/mL CB1 SFTSV strain (genotype B) and incubated with (A, E) rN-based ELISA positive or (B, F) rGn based ELISA positive or (C, G) both rN-based ELISA and rGn based ELISA positive, or (D, H) both rNELISA and rGn ELISA negative deer sera (1:20 dilutions) and fluorescein-labeled deer IgG was used as the secondary antibody. The bar on the bottom right of each image represents the scale of 20 μm.