Fig 2: CsA inhibits the pro-inflammatory effects of CyPA after severe SFTSV infection. Normal human PBMCs (A) and THP-1 cells (B) were divided into blank group, SFTSV-infected (MOI = 0.5) 12 h group, 4 μM CsA pretreatment for 12 h, and SFTSV-infected 12 h (MOI = 0.5) group. The expression of CyPA in the supernatant of cells in each group was measured by ELISA. (C) THP-1 cells were divided into blank group, SFTSV-infected (MOI = 0.5) 12 h group, CsA pretreatment at different concentrations for 12 h, and SFTSV-infected 12 h (MOI = 0.5) group. The total protein in each group of cells was extracted to detect MAPK pathway activation by Western blot. The mRNA levels of IL-6, IL-1β, and TNF-α were measured by qRT-PCR in PBMCs (D) and THP-1 (E). The protein levels of IL-6, IL-1β, and TNF-α in the supernatants of PBMCS (F–H) and THP-1 (I–K) were correspondingly collected and measured by ELISA. (A,B,D–K). The values shown represent the mean ± standard error of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (C) One representative experiment is shown.

Fig 3: Treatment of SFTSV-infected IFNAR-/- mice with CsA. (A) Mice in untreated and CsA-treated groups were inoculated s.c. with 1.0 × 106 TCID50 of SFTSV, while the mock mice were inoculated s.c. with 100 µl DMEM. In the CsA-treated group, three mice were treated with 10 mg/kg/day CsA for five consecutive days. Survival curves were determined using GraphPad Prism7. (B) The relative weight is shown as means with standard deviations. (C) SFTSV RNA levels in plasma samples collected at 1,3, or 5 days post-infection as determined by qRT-PCR. (D) The levels of CyPA in plasma samples were detected by ELISA. (E–G) The protein levels of proinflammatory cytokines in plasma samples as detected by ELISA. Significance was determined relative to the untreated group: *p < 0.05, **p < 0.01, ***p < 0.001, NS, no significance.

Fig 4: Extracellular CyPA binds to CD147 to activate the MAPK pathway in SFTSV-infected THP-1 cells. (A) The expression of CyPA mRNA as detected by qRT-PCR in THP-1 cells transfected with the sh-CyPA, compared with NC cells. (B) The CyPA protein level as detected by Western blot in sh-CyPA compared with NC cells. (C) The gray value analysis of (B). (D) Western blot analysis of the indicated proteins in SFTSV-infected THP-1 lysates. (E) WB analysis of the indicated proteins in SFTSV-infected sh-CyPA and NC cell lysates at different times. (F) THP-1 cells were divided into blank group, AC-73 treated 4 h group, SFTSV or hrCyPA treated 0.5 h group, and co-treatment groups. Total cell lysates of each group were extracted for WB. (A,C) The values shown indicate the mean ± standard error of three independent experiments. **p < 0.01. (D–F) One representative experiment is shown.

Fig 5: CyPA promotes the replication of SFTSV in THP-1 cells. In the experimental group, THP-1 cells were pretreated with hr-CyPA for 2 h and then infected with SFTSV for 48 h, after which the expression of SFTSV in the supernatant (A) and intracellular fluid (B) was determined. In the experimental group, THP-1 cells were infected with SFTSV for 2 h and then treated with hr-CyPA for 48 h, and the expression of SFTSV in the supernatant (C) and cells (D) was measured. In the experimental group, THP-1 cells were infected with SFTSV for 48 h after 2 h CsA pretreatment, and the expression of SFTSV in the supernatant (E) and intracellular fluid (F) was determined. In the experimental group, THP-1 cells were treated with CsA for 48 h after 2 h of SFTSV infection, and the expression of SFTSV in the supernatant (G) and cells (H) was measured. The values represent the mean ± standard error of three independent experiments. **p < 0.01, ***p < 0.001.