Detection of the antiviral activity of epicatechin isolated from Salacia crassifolia (Celastraceae) against Mayaro virus based on protein C homology modelling and virtual screening.

Mayaro fever, caused by Mayaro virus (MAYV) is a sub-lethal disease with symptoms that are easily confused with those of dengue fever, except for polyarthralgia, which may culminate in physical incapacitation. Recently, outbreaks of MAYV have been documented in metropolitan areas, and to date, there is no therapy or vaccine available. Moreover, there is no information regarding the three-dimensional structure of the viral proteins of MAYV, which is important in the search for antivirals. In this work, we constructed a three-dimensional model of protein C of MAYV by homology modelling, and this was employed in a manner similar to that of receptors in virtual screening studies to evaluate 590 molecules as prospective antiviral agents. In vitro bioassays were utilized to confirm the potential antiviral activity of the flavonoid epicatechin isolated from Salacia crassifolia (Celastraceae). The virtual screening showed that six flavonoids were promising ligands for protein C. The bioassays showed potent antiviral action of epicatechin, which protected the cells from almost all of the effects of viral infection. An effective concentration (EC50) of 0.247 µmol/mL was observed with a selectivity index (SI) of 7. The cytotoxicity assay showed that epicatechin has low toxicity, with a 50% cytotoxic concentration (CC50) greater than 1.723 µmol/mL. Epicatechin was found to be twice as potent as the reference antiviral ribavirin. Furthermore, a replication kinetics assay showed a strong inhibitory effect of epicatechin on MAYV growth, with a reduction of at least four logs in virus production. Our results indicate that epicatechin is a promising candidate for further testing as an antiviral agent against Mayaro virus and other alphaviruses.

A mitogenic signal triggered at an early stage of vaccinia virus infection: implication of MEK/ERK and protein kinase A in virus multiplication.

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.