Antiviral Activity of Selective Estrogen Receptor Modulators against Severe Fever with Thrombocytopenia Syndrome Virus In Vitro and In Vivo

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV), also known as the Dabie Banda virus, is an emerging tick-borne Bunyavirus that causes severe fever with thrombocytopenia syndrome (SFTS). Currently, symptomatic treatment and antiviral therapy with ribavirin and favipiravir are used in clinical management. However, their therapeutical efficacy is hardly satisfactory in patients with high viral load. In this study, we explored the antiviral effects of selective estrogen receptor modulators (SERMs) on SFTSV infection and the antiviral mechanisms of a representative SERM, bazedoxifene acetate (BZA). Our data show that SERMs potently inhibited SFTSV-induced cytopathic effect (CPE), the proliferation of infectious viral particles, and viral RNA replication and that BZA effectively protected mice from lethal viral challenge. The mode of action analysis reveals that BZA exerts antiviral effects during the post-entry stage of SFTSV infection. The transcriptome analysis reveals that GRASLND and CYP1A1 were upregulated, while TMEM45B and TXNIP were downregulated. Our findings suggest that SERMs have the potential to be used in the treatment of SFTSV infection.

Keywords: antiviral; bazedoxifene acetate; selective estrogen receptor modulator; severe fever with thrombocytopenia syndrome virus.

Figure 1

Selective estrogen receptor modulators (SERMs) inhibit severe fever with thrombocytopenia syndrome virus in the Huh7 cell line. The half-maximal effect concentration (EC₅₀) and concentration of cytotoxicity 50% (CC₅₀) of SERMs were calculated. Huh7 cell lines were co-cultured with gradient-diluted compounds and 100× TCID₅₀ severe fever with thrombocytopenia syndrome virus for 5–7 days. The left or right y-axes represent the mean % CPE inhibition or cytotoxicity of the drug, respectively. Data were obtained from at least three independent tests. As shown in the figure, (A–H) represents eight SERMs, respectively.

Figure 2

Selective estrogen receptor modulators significantly inhibit severe fever with thrombocytopenia syndrome virus RNA replication in vitro (quantitative real-time PCR). Huh7 cells were incubated with concentrations of test compounds or vehicle and then inoculated with severe fever with thrombocytopenia syndrome virus at an MOI of 0.1 for 48 h at 37 °C. The cells were treated with TRIzol reagent before quantitative real-time PCR assay. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, no significance.

Figure 3

Inhibition effect of selective estrogen receptor modulators on the production of infectious severe fever with thrombocytopenia syndrome virus progeny virions in Huh7 cells. Cells were incubated with different compounds or vehicle (DMSO, dissolvent of the drugs) and inoculated with severe fever with thrombocytopenia syndrome virus at an MOI of 0.1 for 48 h at 37 °C. The supernatant was collected, and infectious viral particles were quantified with PFU assay. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Bazedoxifene acetate (BZA) reduced severe fever with thrombocytopenia syndrome virus (SFTSV) viremia. Blood viral loads in SFTSV-infected Balb/c mice (A) and A129 mice (B) treated with BZA or vehicle were measured by quantitative real-time PCR. *** p < 0.001, **** p < 0.0001; ns, no significance.

Figure 5

Bazedoxifene acetate (BZA) protected against severe fever with thrombocytopenia syndrome virus (SFTSV) challenge in vivo. (A,D) Schematic diagram of mice experiments. One-day-old ICR suckling mice were challenged with 1.25 × 10⁶ PFU SFTSV per mouse and administered with the indicated doses of BZA intraperitoneally (B,C) or by oral gavage to mother mice (E,F). The daily survival rate and changes in body weight of the mice were monitored for 21 days. Survival data were analyzed with a log-rank test. The data of weight curve were analyzed with unpaired, two-tailed t-tests. * p < 0.05, ** p < 0.01; ns, no significance.

Figure 6

Bazedoxifene acetate interferes with severe fever with thrombocytopenia syndrome virus post-entry stage. (A) Scheme of time-of-drug-addition assay. Huh7 cells were seeded and cultured overnight. The virus (MOI = 0.1) was added at 0 h, and drugs were added at indicated time periods (I–IV) to inhibit different stages of the viral life cycle. The polymerase inhibitor T-705 was used as the positive control. (B,C) Intracellular viral RNA or infectious viral titers were quantified by quantitative real-time PCR or PFU, respectively, at 24 hpi. The percentage infection rate was calculated as the “drug-treated group/the virus group”. ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, no significance.

Figure 7

Transcriptome analysis of bazedoxifene acetate (BZA) against severe fever with thrombocytopenia syndrome virus (SFTSV). Huh7 cells were inoculated into 12-well plates at 1 × 10⁵ cells/well and cultured overnight. Subsequently, cells were infected with 0.1 MOI of SFTSV with the presence of 3 μM of BZA. At 24 hpi, cells were harvested to extract total RNA and subjected to transcriptome analysis. (A) The common and unique differentially expressed genes were identified by a Venn diagram. (B) Minus-versus-add plot identified the complex upregulation and downregulation. (C) The number of differentially expressed genes (DEGs).

Figure 8

Top enriched GO annotation analysis (A) and KEGG enrichment analysis (B) for differentially expressed genes that were upregulated by severe fever with thrombocytopenia syndrome virus infection and restored by bazedoxifene acetate (BZA) treatment. (C) The heatmap of key DEGs related to severe fever with thrombocytopenia syndrome virus infection and BZA intervention were visualized. The color shows the fold change of detected genes.