Discordant Activity of Kaempferol Towards Dengue Virus and Japanese Encephalitis Virus

Abstract

Kaempferol, a plant-derived flavonoid, has been reported to have activity against Japanese encephalitis virus (JEV) in BHK-21 cells. To determine the broader utility of this compound, we initially evaluated the activity of kaempferol against JEV and dengue virus (DENV) in HEK293T/17 cells. Results showed no significant antiviral activity against either virus. We subsequently investigated the activity of kaempferol against both JEV and DENV in BHK-21 cells. Results showed a significant inhibition of JEV infection but, surprisingly, a significant enhancement of DENV infection. The effect of kaempferol on both host protein expression and transcription was investigated and both transcriptional and translational inhibitory effects were observed, although a more marked effect was observed on host cell protein expression. Markedly, while GRP78 was increased in DENV infected cells treated with kaempferol, it was not increased in JEV infected cells treated with kaempferol. These results show that cellular alteration induced by one compound can have opposite effects on viruses from the same family, suggesting the presence of distinct replication strategies for these two viruses.

Keywords: Japanese encephalitis virus; Kaempferol; antiviral activity; dengue virus.

Figure 1

Cytotoxicity of kaempferol. The cytotoxicity of kaempferol was evaluated by using MTT assay. Results are presented as percentage of cell viability from four replicates at 24 h post treatment. (A) HEK293T/17 cells and (B) BHK-21 cells were treated with various concentrations of kaempferol in parallel with the corresponding percentage of DMSO. Negative (10% FBS in DMEM) and positive (10% EtOH) controls were included. Error bars represent mean ± SD *; p value < 0.05, **; p value < 0.01 and ***; p value < 0.001. All statistics were determined by comparison with the DMSO control.

Figure 2

Analysis of direct virucidal activity against dengue virus 2 (DENV 2) and Japanese encephalitis virus (JEV). Either DENV 2 or JEV stock virus were directly incubated with various concentrations of kaempferol or corresponding concentrations of DMSO at 37 °C for 1 h. Subsequently, viral titer in pfu/ml was determined by standard plaque assay. Experiment was performed independently in triplicate with duplicate plaque assay. Error bar shows mean ± SD.

Figure 3

Screening of anti-viral activity of kaempferol against DENV 2 and JEV in HEK293T/17 cells. HEK293T/17 cells were infected with either DENV 2 at MOI 5 or JEV at MOI 2. The infected cells were incubated in the presence or absence of various concentrations of kaempferol or parallel concentrations of DMSO vehicle for 24 h. The percentage infection of DENV 2 (A) and JEV (C) were analysed by flow cytometry. The viral titer in the supernatants of DENV 2 (B) and JEV (D) were determined by standard plaque assay. Experiments were undertaken independently in triplicate with duplicate standard plaque assay. Error bars show mean ± SD.

Figure 4

Screening of anti-viral activity of kaempferol against DENV 2 and JEV in BHK-21 cells. BHK-21 cells were infected with either DENV 2 at MOI 5 or JEV at MOI 2. The infected cells were incubated in the presence or absence of various concentrations of kaempferol or parallel concentrations of DMSO vehicle for 24 h. The percentage infection of JEV (A) and DENV (C) was determined by flow cytometry. The viral production in the supernatants of JEV (B) and DENV (D) were determined by standard plaque assay. Experiments were undertaken independently in triplicate with duplicate standard plaque assay. Error bars show mean ± SD. **; p value ≤ 0.01, ***; p value ≤ 0.001. All statistics were determined by comparison with the DMSO control.

Figure 5

Evaluation of effects of kaempferol by indirect immunofluorescence assay. BHK-21 cells were infected with DENV 2 (A) or JEV (B). The infected cells were incubated with various concentrations of DMSO or kaempferol for 24 h, after which cells were probed with mouse anti-dengue complex antibody (A) or with a mouse pan specific anti-flavivirus E protein monoclonal antibody (HB112) (B). Cells were counterstained with DAPI before observation under a confocal microscope.

Figure 6

Evaluation of protein expression after kaempferol treatment. BHK-21 cells were treated with different concentrations of kaempferol or DMSO vehicle control for 24 h after which expression of vinculin, GRP78, Hsp70, β-actin and GAPDH was determined by western blotting using appropriate antibodies.

Figure 7

Evaluation of viral protein expression after treatment with kaempferol. BHK-21 cells were mock infected or infected with either DENV 2 at MOI 5 or JEV at MOI 2 followed by 24 h treatment with kaempferol at the indicated concentrations or with corresponding concentrations of DMSO vehicle. The cells were collected at 24 h post treatment and total proteins extracted. Western blot analysis was performed to validate the protein expression of E protein and NS1 in DENV 2 infection (A) and JEV E protein in JEV infection (B). Hsp70 was used as an internal protein loading control. Experiments were undertaken independently in triplicate.

Figure 8

Effect of kaempferol treatment on gene transcription in BHK-21 cells. BHK-21 cells were treated with different concentrations of kaempferol or DMSO vehicle control for 24 h after which expression of β-actin, GAPDH and GRP78 was determined by semi-quantitative PCR (A). The band intensity was quantitated using ImageJ analysis software and analysed by GraphPad Prism7 program. The relative expression level of all transcripts was quantitated against the average value and the analysis is presented as bar graph (B). Experiment was undertaken independently in triplicate and data was normalized against the average signal for all conditions. Error bars represent SD, *; p value ≤ 0.05, **; p value ≤ 0.01. All statistics were evaluated by comparing between the treated samples and the equivalent DMSO control.

Figure 9

Effect of kaempferol treatment on gene transcription of BHK-21 cells infected with JEV. BHK-21 cells were infected with JEV at MOI 2 and subsequently incubated with varying concentrations of kaempferol or vehicle control for 24 h after which expression of β-actin, GAPDH and GRP78 was determined by semi-quantitative PCR (A). The band intensity was quantitated using ImageJ analysis software and analysed by GraphPad Prism7 program. The relative expression level of all transcripts was quantitated against the average value and the analysis is presented as bar graph (B). Experiment was undertaken independently in triplicate and data was normalized against the average signal for all conditions. Error bars represent SD, *; p value ≤ 0.05, **; p value ≤ 0.01. Statistical significance was derived from comparison with the control or with the corresponding DMSO control where indicated.

Figure 10

Effect of kaempferol treatment on gene transcription of BHK-21 cells infected with DENV 2. BHK-21 cells were infected with DENV 2 at MOI 5 and subsequently incubated with varying concentrations of kaempferol or vehicle control for 24 h after which expression of β-actin, GAPDH and GRP78 was determined by semi-quantitative PCR (A). The band intensity was quantitated using ImageJ analysis software and analysed by GraphPad Prism7 program. The relative expression level of all transcripts was quantitated against the average value and the analysis is presented as bar graph (B). Experiment was undertaken independently in triplicate and data was normalized against the average signal for all conditions. Error bars represent SD, *; p value ≤ 0.05, **; p value ≤ 0.01. Statistical significance was derived from comparison with DENV infection or the DMSO control where indicated.

Figure 11

Effect of kaempferol treatment on GRP78 expression. BHK-21 cells were mock infected or infected with JEV or DENV 2 and treated with 100 μM kaempferol, DMSO vehicle or not treated (control) for 24 h. The cells were collected at 24 h post treatment and total proteins were extracted. Western blot analysis was performed to determine the expression of GRP78. Hsp70 was used as an internal protein loading control. Experiments were undertaken independently in triplicate. Error bars represent SD. *; p value ≤ 0.05, **; p value ≤ 0.01.