In Vitro Antiviral Activity of Rhodiola crenulata Extract against Zika Virus and Japanese Encephalitis Virus: Viral Binding and Stability

Abstract

Zika virus (ZIKV) and Japanese encephalitis virus (JEV) can cause permanent neurological damage and death, yet no approved drugs exist for these infections. Rhodiola crenulate, an herb used in traditional Chinese medicine for its antioxidation and antifatigue properties, was studied for its antiviral activity against ZIKV and JEV in vitro. The cytotoxicity of Rhodiola crenulata extract (RCE) was evaluated using the CCK-8 reagent. Antiviral effects of RCE were assessed in ZIKV-infected or JEV-infected Vero cells via quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, fluorescent focus assay (FFA), and immunofluorescence assay (IFA). The cell-free antiviral effects of RCE were evaluated using an inactivation assay. To determine the stage of the viral life cycle affected by RCE, time-of-addition, binding, and entry assays were conducted. Three bioactive constituents of RCE (salidroside, tyrosol, and gallic acid) were tested for antiviral activity. RCE exhibited dose-dependent anti-ZIKV and anti-JEV activities at non-cytotoxic concentrations, which were likely achieved by disrupting viral binding and stability. Gallic acid exhibited antiviral activity against ZIKV and JEV. Our findings indicate that RCE disrupts viral binding and stability, presenting a potential strategy to treat ZIKV and JEV infections.

Keywords: Japanese encephalitis virus; Rhodiola crenulata; Zika virus; antiviral agent; gallic acid; salidroside.

Figure 1

Photograph of plant Rhodiola crenulata and chemical structures of target compounds. (A) Rhodiola crenulata. (B) Salidroside. (C) Tyrosol. (D) Gallic acid.

Figure 2

Antiviral activity of Rhodiola crenulata extract (RCE) on Zika virus (ZIKV). Infected Vero cells (MOI = 0.01) were incubated in 12-well plates with various concentrations of RCE for 48 h: (A) Intracellular ZIKV RNA levels were determined using qRT-PCR. (B) Extracellular progeny yields were determined using fluorescent focus assay (FFA). (C) The inhibitory effects on the infection ability were determined using immunofluorescence assay (IFA). Magnification power of the microscope was 200 times. Green fluorescence indicates virus-infected cells. (D) ZIKV Env protein expression was measured using Western blot analysis. The data are presented as the mean ± SD of experiments conducted in triplicate. Statistical significance was assessed via one-way ANOVA versus the virus control group: * indicates p < 0.05.

Figure 3

Antiviral activity of Rhodiola crenulata extract (RCE) on Japanese encephalitis virus (JEV). Vero cells were inoculated with JEV (MOI = 0.02) in 12-well plates with indicated concentrations of RCE for 48 h: (A) Intracellular JEV RNA levels were determined using qRT-PCR. (B) Extracellular progeny yields were determined using fluorescent focus assay (FFA). (C) The inhibitory effects on infection were determined using immunofluorescence assay (IFA). Magnification power of the microscope was 200 times. Green fluorescence indicates virus-infected cells. (D) JEV NS3 protein expression was determined via Western blot analysis. The data are presented as the mean ± SD of experiments conducted in triplicate. Statistical significance was assessed via one-way ANOVA versus the virus control group: * indicates p < 0.05.

Figure 4

Time-of-addition assay results. (A) Timeline of time-of-addition assays. The red line indicates the viral infection, the blue line indicates the Rhodiola crenulata extract (RCE) administration period, and the dotted line represents the incubation period. Infected cells (MOI = 0.5) were treated with RCE in 12-well plates at various time points relative to viral infection. After a 24 h incubation. (B) Zika virus (ZIKV) or (C) Japanese encephalitis virus (JEV) RNA levels were measured using qRT-PCR. Dose-inhibition assays involved the simultaneous exposure of cells to the drug and virus at increasing concentrations (MOI = 0.5) for 2 h (absorption period), after which the supernatant was discarded and the cells were washed twice with PBS. (D) ZIKV or (E) JEV RNA expression was measured using qRT-PCR after incubation for 24 h. The data are presented as the mean ± SD of experiments conducted in triplicate. Statistical significance was assessed via one-way ANOVA versus the virus control group: * indicates p < 0.05.

Figure 5

Binding assays for (A) Zika virus (ZIKV) and (B) Japanese encephalitis virus (JEV). Entry assays for (C) ZIKV and (D) JEV. The data are presented as the mean ± SD of experiments conducted in triplicate. Statistical significance was assessed via one-way ANOVA versus the virus control group: * indicates p < 0.05.

Figure 6

Inactivation assay of Rhodiola crenulata extract (RCE) on Zika virus (ZIKV) and Japanese encephalitis virus (JEV), which involved incubating RCE with (A) ZIKV (2 × 10⁷ FFU) or (B) JEV (2 × 10⁷ FFU) at 37 °C for 2 h. Virion stability was evaluated using FFA. The data are presented as the mean ± SD of experiments conducted in triplicate. Statistical significance was assessed via one-way ANOVA versus the virus control group: * indicates p < 0.05.

Figure 7

Antiviral effects of Rhodiola crenulata extract (RCE) constituents, salidroside, tyrosol, and gallic acid on (A–C) Zika virus (ZIKV) and (D–F) Japanese encephalitis virus (JEV). After treating virus-infected Vero cells (ZIKV MOI = 0.01; JEV MOI = 0.02) with each of the constituent compounds for 24 h, viral RNA levels were measured using qRT-PCR. Statistical significance was assessed via one-way ANOVA versus the virus control group: * indicates p < 0.05.

Figure 8

Antiviral activity of gallic acid on Zika virus (ZIKV) and Japanese encephalitis virus (JEV). Infected Vero cells were inoculated in 12-well plates with indicated concentrations of gallic acid for 48 h. Magnification power of the microscope was 200 times. The inhibitory effects on infection were determined using immunofluorescence assay (IFA). Green fluorescence indicates virus-infected cells. IFA results for (A) ZIKV and (B) JEV.