Resveratrol reduced the cell death caused by MERS-CoV

To investigate the anti-MERS-CoV effect of resveratrol, we directly treated MERS-CoV infected Vero E6 cells with different concentrations of resveratrol. Cells were infected with MERS-CoV at M.O.I. of 0.1. After 48 hours, we imaged the cellular morphology via microscopy and measured cell proliferation by MTT assay, cell viability by neutral red uptake (NRU) assay, and cytotoxicity levels by lactate dehydrogenase (LDH) assay. Resveratrol at 250 and 125 μM seems to alleviate the monolayer destruction of the Vero E6 cells infected by MERS-CoV (Fig. 1). Results from MTT assays (Fig. 1a) and NRU assays (Fig. 1b) showed that resveratrol can reduce the cell death induced by MERS-CoV infection in the concentration range from 250–125 μM. Also, resveratrol-treated groups revealed less cytotoxicity by LDH assay after MERS-CoV infection (Fig. 1c) and the cytotoxicity profiles correlated well with that of cell proliferation and cell viability assays. To determine if the observed antiviral effect by resveratrol was due to its cytotoxicity, we performed LDH assay for resveratrol treatment only. Figure 1d showed that resveratrol caused limited cytotoxicity to Vero E6 cells. Even at the highest concentration of 250 μM, the cytotoxicity was no more than 25%. In consistence, the cytotoxicity of MERS-CoV-infected cells was reduced by resveratrol treatment (250 μM) to approximately 25% (Fig. 1c). Therefore, we conclude that resveratrol reduced the cell death caused by MERS-CoV infection.

Fig. 1 Resveratrol reduced the cell death caused by MERS-CoV infection. Vero E6 cells were infected by MERS-CoV with M.O.I of 0.1 and treated with resveratrol for 48 hours. The level of cell viability was determined by (a) MTS assay (b) neutral red uptake assay, and (c) LDH assay. d Resveratrol itself caused limited cytotoxicity to Vero cells by LDH assay Full size image

Resveratrol reduced the RNA expression and viral yield of MERS-CoV

To determine if resveratrol directly inhibits MERS-CoV infection, we assessed the effects of resveratrol on MERS-CoV viral production at the RNA level. We collected cell samples with or without viral infection after resveratrol treatments at 24 and 48 hours post-infection (h.p.i). Extracted total RNA was subjected to quantitative real-time PCR to compare the relative MERS-CoV RNA levels. Shown in Fig. 2a, the MERS-CoV RNA levels in resveratrol-treated cells at concentrations of 250, 200, 150, 62.5, and 31.25 μM were significantly lower than in MERS-CoV-infected cells at 24 h.p.i. However, the inhibitory effects of low concentrations of resveratrol, including 62.5 and 31.25 μM, diminished at 48 h.p.i. This data suggests that resveratrol treatment suppressed MERS-CoV RNA replication, although it requires relatively high concentrations of resveratrol to deliver persisted antiviral effects.

Fig. 2 Resveratrol decreased MERS-CoV RNA and viral plaques. a MERS RNA level was monitored at 24 and 48 h.p.i. by real-time PCR after resveratrol treatment. Relative RNA levels were determined by comparing MERS only groups at each time point. GAPDH RNA was used as an internal control. b Quantification of plaque reduction assay of MERS-CoV titer after treated with resveratrol from 250 μM to 31.25 μM for 48 hours Full size image

Next, we determined the infectious titer of MERS-CoV after resveratrol treatments by plaque reduction assays. Data in Fig. 2b showed that MERS titers were significantly reduced by resveratrol treatment at 250, 200 and 150 μM respectively, at 48 h.p.i. This result is consistent with those obtained from the quantitative real-time PCR at 48 h.p.i. In summary, these findings indicate that treatment of cells with resveratrol reduced the MERS-Cov RNA levels and infectious titers, which presumably accounted for the observed decrease in cell death.

Resveratrol inhibited existing MERS-CoV infection

Previous studies have shown that resveratrol exerted antiviral activities by blocking NF-κB pathway [22, 30], suggesting resveratrol has a broad spectrum of antiviral effects by down-regulating inflammatory signaling transduction. To determine whether resveratrol inhibits the entry or a post-entry step of MERS-Cov infection, we added resveratrol together with MERS-CoV immediately for 3 hours or after the infection has been initiated (Fig. 3a and b). We measured the cell proliferation (Fig. 3c and d) and determined the viral titers in the supernatants (Fig. 3e). The results demonstrated that even when resveratrol was given after MERS-CoV infection, it still reduced the viral titer. The same observations were made when cell proliferations and viral titers were measured, suggesting that resveratrol inhibits MERS-CoV infection after entry.

Fig. 3 Resveratrol inhibited the existing MERS infection. Resveratrol and MERS-CoV were simultaneously added into cells for first 3 hours before removing virus and adding new resveratrol for the rest incubating time (a). MERS-CoV infected Vero cells for first 3 hours before washed out and treated with resveratrol (b). The trends of cell proliferation by MTT assays (c and d) and MERS-CoV titers by plaque assays (e) were similar, revealing resveratrol inhibited MERS viral yield even if existing MERS infection Full size image

Resveratrol inhibited MERS-CoV nucleocapsid expression

To corroborate our findings, we stained nuclecapsid (N) protein of MERS-CoV after resveratrol treatments at 24 h.p.i and visualized the N protein distribution by confocal microscopy. Images shown in Fig. 4a demonstrated that 250 μM of resveratrol eliminated the N protein fluorescent signal compared to control groups, while 150 μM of resveratrol exhibited a limited decreasing of N protein signal. In order to elucidate whether the strength of N protein signal was correlated to the concentration of resveratrol, we performed intracellular staining of N protein in cells cultured in multiple well plates. MERS-infected Vero E6 cells were fixed and permeablized to facilitate anti-N primary antibody to access the target protein and quantified the strength of fluorescence by Li-Cor imaging system (Fig. 4b and c). The results showed that resveratrol remarkably inhibited MERS nucleocapsid protein translation in a dose-dependent manner, especially in the concentration of 250 to 125 μM.

Fig. 4 Resveratrol reduced nucleocapsid expression of MERS-CoV. Vero E6 cells were infected by MERS-CoV (M.O.I. 0.1) and treated with resveratrol for 24 hours followed by 4% paraformaldehyde fixation for immunofluorescent assays. a Nucleocapsid expressions were examined with confocal microscope at 680× magnification. DAPI was used for nucleus staining. b Intracellular staining of MERS nucleocapsid expressions were visualized by Odyssey® CLx Imaging system. c Quantification results of fluorescent intensities of MERS nucleocapsid proteins were determined by Odyssey® CLx Imaging software Full size image

Resveratrol inhibited Caspase 3 cleavage induced by MERS-CoV infection

Apoptosis in different tissues during MERS-CoV infection has been widely documented [31, 32]. We therefore tested whether resveratrol is able to reduce the apoptosis induced by MERS-CoV. Due to the cleavage of Caspase 3, an indicator of apoptosis, was reportedly elevated during MERS-CoV infection [33], we collected the cell lysates after MERS-CoV infection and resveratrol treatments at 24 and 48 h.p.i. and conducted western blotting to measure the Caspase 3 cleavage levels. The results (Fig. 5) show that the protein expression of the cleaved Caspase 3 significantly increased after MERS-CoV infection, confirming that MERS-CoV could cause cellular apoptosis. Interestingly, when resveratrol was added, the levels of Caspase 3 cleavage decreased. As the concentration of resveratrol went higher, the levels of Caspase 3 cleavage decreased in a dose-dependent manner (Fig. 5c). Our results suggest that resveratrol reduced the MERS-CoV-mediated apoptosis. Notably, resveratrol at 250 μM did not lower the Caspase 3 cleavage to the same level, as did 200 μM (Fig. 5a, c). Given to the cytotoxicity of resveratrol itself, this result is expected.

Fig. 5 Resveratrol reduced MERS-induced cell apoptosis. Vero E6 cells were infected by MERS-CoV at M.O.I. of 0.1 and treated with resveratrol for 24 and 48 hours before collecting protein lysates. a Protein expression levels of MERS nucleocapsid and cleaved caspase 3 were evaluated by western blotting. GAPDH was used as a loading control. b and c Protein expression levels were quantified and then normalized with GAPDH expression. MERS nucleocapsid as well as cleaved Caspase 3 expressions were reduced by resveratrol in a dose-dependent manner Full size image

Consecutively administration of resveratrol at lower concentrations inhibited MERS infection

From the qPCR (Fig. 2a) and western blotting results (Fig. 5), we noticed that resveratrol at 62.5 μM and below appeared to inhibit MERS-CoV within 24 hours but then lost effects at 48 h.p.i. The loss of inhibitory effects at 48 hrs could due to the degradation of resveratrol in the media after such long period of incubation. Given the high dosages of resveratrol still has some cytotoxicity (Fig. 1e), lower dosages are more desirable to treat MERS-infected patients clinically. To explore the possibility of utilization of resveratrol at lower dosages, we added resveratrol consecutively (every 24 hrs) to MERS-infected cells at lower concentrations and evaluated the cell proliferation, cell viability, and cytotoxicity (Fig. 6). Interestingly, consecutive addition of resveratrol at 62.5 μM but not 31.25 μM or below partially rescued MERS-Cov-induced cell death (Fig. 6a-c) and lowered the production of infectious MERS-CoV by approximate tenfold (Fig. 6d), indicating resveratrol can inhibit MERS-CoV by administrating at lower dosages if given consecutively.

Fig. 6 Consecutively resveratrol administration inhibited MERS infection in a lower dosage. MERS-infected Vero E6 cells were treated with resveratrol in lower dosages every 24 hours. The cell proliferation by MTT assay (a), cell viability by NRU assay (b), cytotoxicity by LDH assay (c), and plaque assay (d) were utilized to measure the cell survival after 48 hours of MERS infection at M.O.I. of 0.1 Full size image

Resveratrol exhibited extended antiviral activities

Furthermore, we tested the effects of resveratrol on another emerging positive-sense RNA virus, chikungunya virus. As shown in Fig. 7, resveratrol not only inhibited MERS-CoV viral production but also reduced the production of chikungunya virus at concentrations of 250 and 125 μM. Altogether, our data suggests that resveratrol might be a lead candidate for further pre-clinical assessments of antiviral activity for MERS-CoV and additional emerging RNA viruses.