Effective inhibition of MERS-CoV infection by resveratrol

Abstract

Background: Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that causes severe morbidity and mortality. Up to date, there is no approved or licensed vaccine or antiviral medicines can be used to treat MERS-CoV-infected patients. Here, we analyzed the antiviral activities of resveratrol, a natural compound found in grape seeds and skin and in red wine, against MERS-CoV infection.

Methods: We performed MTT and neutral red uptake assays to assess the survival rates of MERS-infected Vero E6 cells. In addition, quantitative PCR, western blotting, and immunofluorescent assays determined the intracellular viral RNA and protein expression. For viral productivity, we utilized plaque assays to confirm the antiviral properties of resveratrol against MERS-CoV.

Results: Resveratrol significantly inhibited MERS-CoV infection and prolonged cellular survival after virus infection. We also found that the expression of nucleocapsid (N) protein essential for MERS-CoV replication was decreased after resveratrol treatment. Furthermore, resveratrol down-regulated the apoptosis induced by MERS-CoV in vitro. By consecutive administration of resveratrol, we were able to reduce the concentration of resveratrol while achieving inhibitory effectiveness against MERS-CoV.

Conclusion: In this study, we first demonstrated that resveratrol is a potent anti-MERS agent in vitro. We perceive that resveratrol can be a potential antiviral agent against MERS-CoV infection in the near future.

Keywords: Inhibition; MERS-CoV; MERS-CoV virus infection; Middle East Respiratory Syndrome Virus; Resveratrol.

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

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

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

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

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

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

Fig. 7

Resveratrol showed antiviral activity against chikungunya infection. Vero E6 cells were infected by chikungunya virus at M.O.I. of 0.1 and treated with resveratrol for 48 hours. The quantitative viral titers of chikungunya by plaque assay were decreased by resveratrol in 250 μM and 125 μM concentrations