Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality

Abstract

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne infectious disease caused by a novel phlebovirus (SFTS virus, SFTSV), was listed among the top 10 priority infectious diseases by the World Health Organization due to its high fatality of 12%-50% and possibility of pandemic transmission. Currently, effective anti-SFTSV intervention remains unavailable. Here, by screening a library of FDA-approved drugs, we found that benidipine hydrochloride, a calcium channel blocker (CCB), inhibited SFTSV replication in vitro. Benidipine hydrochloride was revealed to inhibit virus infection through impairing virus internalization and genome replication. Further experiments showed that a broad panel of CCBs, including nifedipine, inhibited SFTSV infection. The anti-SFTSV effect of these two CCBs was further analyzed in a humanized mouse model in which CCB treatment resulted in reduced viral load and decreased fatality rate. Importantly, by performing a retrospective clinical investigation on a large cohort of 2087 SFTS patients, we revealed that nifedipine administration enhanced virus clearance, improved clinical recovery, and remarkably reduced the case fatality rate by >5-fold. These findings are highly valuable for developing potential host-oriented therapeutics for SFTS and other lethal acute viral infections known to be inhibited by CCBs in vitro.

Fig. 1

Cell-based high-throughput screening of FDA-approved drug library identified benidipine hydrochloride as an inhibitor of SFTSV replication. a Cell-based high-throughput screening to identify inhibitors of SFTSV replication. b Analysis of screen reproducibility. Dots represent the inhibition rate (left) and cell survive rate (right) for each drug from two replicates of the screen. Red dots indicated 14 drugs that inhibited virus infection (inhibition rate > 70%) without causing significant cytotoxicity (survive rate > 70%). c Dose-dependent inhibitory effects of the seven selected compounds analyzed in Vero cells. Vero cell monolayers were treated with each drug at the indicated concentrations for 1 h, and then inoculated with SFTSV at MOI of 1. At 36 hpi, cells were fixed, and the infected cells were detected through immunofluorescence analysis using antibody against NP protein. The chemical structure of each compound is displayed. d Survival rate analysis of benidipine hydrochloride treatment in Vero cells. CC₅₀: 50% cytotoxic concentration. e Dose-dependent inhibition effects of benidipine hydrochloride on SFTSV infection. IC₅₀: 50% inhibitory concentration. f Inhibition effect of benidipine hydrochloride on the production of infectious SFTSV progeny virions in Vero cells. Vero cells were treated with serial concentrations of benidipine hydrochloride and infected with SFTSV, and virus titer in the supernatant at 16 hpi was determined. g Inhibition effect of benidipine hydrochloride on the production of infectious SFTSV progeny virions in Huh7 cells. Experiment in Huh7 cells was performed as described in f. h Inhibition effect of benidipine hydrochloride on intracellular SFTSV vRNA level in Huh7 cells. Huh7 cells were treated with serial concentrations of benidipine hydrochloride and infected with SFTSV. At 48 hpi, intracellular RNA was extracted and the vRNA level of SFTSV was measured with qRT-PCR. Comparison of mean values between benidipine hydrochloride-treated group and vehicle (DMSO) group (f–h) was analyzed by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. R² (c, f–h) was estimated by nonlinear regression model (curve fit)

Fig. 2

In vitro anti-SFTSV mechanism of benidipine hydrochloride. a Effect of benidipine hydrochloride on SFTSV entry. Vero cells were treated with benidipine hydrochloride or Vehicle (DMSO) and then infected with SFTSV. At 1 hpi, benidipine hydrochloride was removed, and relative intracellular vRNA level of SFTSV was measured at 16 hpi. b Effect of benidipine hydrochloride on the internalization of SFTSV. Vero cells were treated with benidipine hydrochloride or vehicle (DMSO) and then infected with SFTSV in the presence of ammonium chloride. At 3 hpi, cell surface-bound virions were removed by tryspin, and relative vRNA level of internalized SFTSV was measured. c Fluorescence microscopy analysis of effect of benidipine hydrochloride on SFTSV internalization. Vero cells were infected with SFTSV at MOI of 20 in the presence of benidipine hydrochloride (20 μM) or vehicle (DMSO) for 1 h. Cells were washed with PBS, briefly treated with trypsin and then labeled with NP antibody, and cross sections of the middle of the cells were imaged. Representative images of drug- or vehicle (DMSO)-treated cells were shown. White lines indicate cell boundaries. d Quantification of intracellular viral particles of each cell in c was performed and analyzed. Red line indicates median value of each group. n values indicate number of cells analyzed. e Effect of benidipine hydrochloride on SFTSV post-entry events. Vero cells were infected with SFTSV and then benidipine hydrochloride was added at 3 hpi. Relative intracellular vRNA level of SFTSV was measured at 16 hpi. f, g Effect of benidipine hydrochloride on SFTSV MG activity. BSRT7 cells were transfected with SFTSV MG system and vehicle (DMSO) or benidipine hydrochloride was added at the indicated concentration. MG activity was assessed by the percentage of the eGFP-positive cells (f) and the eGFP signal intensity at single-cell level (g). Red lines indicate median values. Comparisons of mean values (a, b, and e) and median values (d, g) between two groups were performed by Student’s t-test and Mann-Whitney test, respectively. Comparison of the percentage of MG activity positive cells between two groups (f) was analyzed by χ² test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

In vitro anti-SFTSV activity of CCBs. a Vero cells were cultured in Ca²⁺-free or normal medium and then infected with SFTSV (MOI = 1). At 16 or 24 hpi, relative intracellular SFTSV vRNA level was measured by qRT-PCR. b Relative intracellular vRNA level of SFTSV in Vero cells upon treatment with BAPTA-AM at 24 hpi. c Intracellular Ca²⁺ concentration in SFTSV-infected cells. Vero cells were infected with SFTSV at the indicated MOI in the presence of benidipine hydrochloride or nifedipine or vehicle (DMSO). Relative intracellular Ca²⁺ level was determined by fluorescence of Fluo-4NW. d Relative intracellular SFTSV NP level in the infected Vero cells. Vero cells were treated with the indicated 14 CCBs or vehicle (DMSO), infected with SFTSV at MOI of 1 and the relative NP level was determined by immunofluorescence with NP antibody at 36 hpi. e Survival rate analysis (left) and dose-dependent inhibition effects (right) of nifedipine treatment in Vero cells. f Relative intracellular SFTSV NP level in SFTSV-infected Huh7 cells. Cells were transfected with siRNA against L-type calcium channel Ca_v1.2 for 48 h and then infected with SFTSV for 48 h, followed by western blot analysis of intracellular NP level. Comparison of mean values (a, b, and d) between two groups was analyzed by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Anti-SFTSV effect of benidipine hydrochloride and nifedipine in mouse models. a, b Spleen (a) or serum (b) viral loads in SFTSV-infected C57BL/6 mice (five for each group) treated with benidipine hydrochloride, nifedipine, or vehicle (0.3% carboxymethyl cellulose solution), were measured by qRT-PCR at 1, 3, 5, and 7 dpi. c Serum viral loads in SFTSV-infected humanized mice treated with benidipine hydrochloride (n = 6), nifedipine (n = 6), or vehicle (0.3% carboxymethyl cellulose solution) (n = 7), were measured by qRT-PCR at 1, 3, 5, 7, 9, 13, and 17 dpi. d Analysis of benidipine hydrochloride and nifedipine treatment on survival probability in humanized mice. The Kaplan–Meier method was used to analyze time-to-event data. The mean value of each group (a–c) was indicated by horizontal line. The significance of the difference between mean values was determined by Student’s t-test. P values < 0.05 were indicated for each comparison

Fig. 5

Clinical effect analysis of nifedipine treatment on SFTS-related fatality and clearance of SFTSV. a Flowcharts of recruitment and grouping of SFTS patients. A total of 2087 patients who had been admitted into the sentinel hospital for SFTS, were included in the retrospective clinical investigation that was performed to evaluate the clinical effect of nifedipine in treating SFTS. By thoroughly reviewing the medical records of these patients, 83 nifedipine-treated, 48 non-nifedipine-treated, and 249 general SFTS patients, were included for comparison. CCBs, calcium channel blocks. b Analysis of nifedipine treatment on survival probability. The Kaplan-Meier method was used to analyze time-to-event data. N_T, the number of nifedipine-treated patients; N_G, the number of general SFTS patients; N_N, the number of non-nifedipine-treated patients. c Analysis of nifedipine treatment effect on the viral loads in SFTS patients. The GEE model was used to analyze sequentially tested viral loads during hospitalization; statistical analysis results were presented in Supplementary information, Table S7. The circle indicates mean value and the dashed line indicates standard deviation. d Analysis of nifedipine treatment effect on the viral clearance in SFTS patients. χ² test was used to compare the frequency difference between two groups, and P values < 0.05 were shown. The circle indicates frequency and the dashed line indicates 95% CI. N_T indicates number of patients in nifedipine-treated group, N_G indicates number of patients in general SFTS group, and N_N indicates number of patients in non-nifedipine group

Fig. 6

Clinical effect analysis of nifedipine treatment on severe complication development and laboratory parameter recovery of SFTS patients. a Left: frequencies of severe complications presented before or at admission; right: frequencies of severe complications developed during hospitalization. Comparisons of severe complications that developed during hospitalization in nefidipine-treated group with those in general SFTS group or in non-nifedipine group were performed by χ² test or Fisher’s exact test. P values < 0.05 were shown. b, c Sequential data on PLT count (b) and LDH level (c) were analyzed by GEE models; statistical analysis results were presented in Supplementary information, Table S7. The circle indicates median value and the dashed line indicates interquartile-range. N_T indicates number of patients in nifedipine-treated group, N_G indicates number of patients in general SFTS group, and N_N indicates number of patients in non-nifedipine group