Antiviral Activity of Resveratrol against Human and Animal Viruses

Resveratrol is a potent polyphenolic compound that is being extensively studied in the amelioration of viral infections both in vitro and in vivo. Its antioxidant effect is mainly elicited through inhibition of important gene pathways like the NF-κβ pathway, while its antiviral effects are associated with inhibitions of viral replication, protein synthesis, gene expression, and nucleic acid synthesis. Although the beneficial roles of resveratrol in several viral diseases have been well documented, a few adverse effects have been reported as well. This review highlights the antiviral mechanisms of resveratrol in human and animal viral infections and how some of these effects are associated with the antioxidant properties of the compound.


Introduction
Resveratrol (RSV) is a naturally occurring polyphenol stilbene found mostly in fermented grapes, mulberry, red wine, and peanuts. It is available in the trans-and cis-isomer forms; however, the cis-resveratrol isomer is unstable and easily transformed into the trans-form when reacted to light. It is insoluble in water but soluble in polar solvents such as ethanol and dimethyl sulfoxide. Resveratrol scavenges for superoxide and hydroxyl in vitro, as well as lipid hydroperoxyl radicals [1]. Previous studies have shown resveratrol to enhance longevity, regulate lipid levels, and act as a prophylactic compound against cancers and related viral infections [2]. It also attenuates superoxide generation in the mitochondria and inhibits mitochondrial dysfunction induced by arachidonic acid [2,3]. The antiviral mechanisms and effects of RSV have been widely studied in a number of viruses which include influenza virus, hepatitis C virus [4], respiratory syncytial virus [5][6][7][8][9], varicella zoster virus [10], Epstein-Barr virus [11,12], herpes simplex virus [13][14][15][16], human immunodeficiency virus [17,18], African swine fever virus, enterovirus, human metapneumonia virus, and duck enteritis virus and in multiple sclerosis, whose animal models can be induced by viral infection. In almost all of these studies, RSV showed remarkable recession of the viral infection with the exception of multiple sclerosis and hepatitis C, where disease progression was worsened following administration of RSV [4,19]. the liver into glucuronides and sulfates [21]. Even though the amount of oral dosing of RSV did not significantly affect its bioavailability in plasma, the type of food consumed and intraindividual differences in metabolism were shown to significantly affect its bioavailability [20,22]. In a related study, the plasma bioavailability of RSV 30 minutes after oral consumption of red wine was only in trace amounts, while moments later RSV glucuronides were systemically abundant for a prolonged time [23]. Recently, research has been focused on developing structured nanoparticles that will enhance the bioavailability of RSV and prolong its release in vivo. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) loaded with RSV were shown to have an entrapment efficiency of 70% and stability lasting for over 2 months. In vitro simulation studies showed a slow sustained release of RSV at both stomach and intestinal pH levels [24]. Similarly, the use of zein-based nanoparticles was reported to enhance the in vivo delivery of RSV in mouse model of endotoxic shock [25].
Resveratrol has been shown to induce apoptosis by upregulating and downregulating numerous genes that are important in cellular function including but not limited to TRAIL-R2/DR5, TRAIL-R2/DR4, p53, Bim, Noxa, PUMA, Bak, Bax, Mcl-1, survivin, Bcl-XL, and Bcl-2. RSV has been shown to inhibit cellular growth at G1 and G1/S phases as well as be an anti-inflammatory mediator by inhibition of nuclear factor-kappa (NF-) activity, procyclooxygenase-2 activity, and prostaglandin production. Furthermore, its actions in delaying the onset of cardiovascular diseases and cancer progression as well as antiviral effects have been widely studied as well [2,20].

Mechanism of Antioxidant Action
Resveratrol (RSV) scavenges for O 2 − and OH − in vitro as well as lipid hydroperoxyl free radicals. One of its most important antioxidant effects is elicited through the inhibition of reactive oxygen species production and glutathione depletion. It also attenuates superoxide generation in the mitochondria and inhibits mitochondrial dysfunction induced by arachidonic acid. These actions have thus resulted in restoration of mitochondrial membrane through mediation of the AMPK-mediated inhibitory phosphorylation of GsK3 downstream of poly(ADP-ribose) polymerase-LK 1 pathway [26,27]. RSV also increased the levels of catalase, superoxide dismutase, and heme-oxygenase-1 in cultured epithelial cells exposed to H 2 O 2 . This shows its remarkable ability in mediating the major antioxidant enzymes involved in the breakdown of H 2 O 2 [28]. Similarly, RSV has been shown to have an 89.1% inhibition of lipid peroxidation of linolenic acid emulsion and has strong scavenging activities against 2,2-diphenyl-1-picrylhydrazyl, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), N,N-dimethyl-pphenylenediamine, O 2 − , and H 2 O 2 . It also has a high reducing power and Fe 2+ chelating activity [3]. However, a recent study showed that the antioxidant effect of RSV is largely dependent on its dose as higher doses were shown to increase ROS production resulting in mitochondrial-dependent death in endothelial cells [29]. It could be thus said that RSV acts as both an antioxidant and a prooxidant depending on the dose administered.

Prophylactic and Therapeutic Uses in Virus Associated Conditions
The antiviral effects of RSV have been demonstrated in a number of pathogenic human and animal viruses. However, in most of these reports, inhibition of virus proliferation was not directly associated with its antioxidant activity but its ability to inhibit viral protein production and gene expression at various levels [41,42].

Influenza Virus. In influenza virus infection, RSV was
shown to actively block nuclear-cytoplasmic translocation of viral ribonucleoproteins in MDCK cells, thus decreasing the expression of late viral proteins related to inhibition of protein kinase C associated pathways. This activity was also found to be unassociated with glutathione-mediated antioxidant activity of the compound [30]. The various mechanisms of viral inhibition exerted by resveratrol on certain viruses are summarized in Table 1.

Epstein-Barr Virus.
In Epstein-Barr virus (EBV) infection, RSV showed an enhanced inhibitory effect on EBV early antigen induction using Raji cells. It was also shown to reduce papilloma production in mouse by 60% after 20 weeks of inoculation [31]. In another study, RSV was shown to dosedependently inhibit EBV lytic cycle by inhibition of transcription genes and proteins, Rta, Zta, and diffused early antigen (EA-D), as well as inhibiting the activity of EBV immediateearly protein: BRLF1 and BZLF1 promoters. This effect was seen to reduce virion production [12]. Similarly, another in vitro study confirmed the previous finding that RSV does inhibit lytic gene expression and viral particle production in a dose-dependent manner. Here its main antiviral mechanism was associated with inhibition of protein synthesis, reduction in ROS production, and inhibition of transcription factors NF-and AP1 [32]. Since EBV is one of the most renowned oncogenic viruses, it is pertinent to study the role of EBV in cellular transformation and cancer progression. RSV was thus shown to prevent transformation of EBV in human B-cells through downregulation of antiapoptotic proteins: Mc1 and survivin. This was also linked to suppression of EBV induced signaling of NF-and STAT-3, as well as miR-155 and miR-34a in EBV infected cells [11].
Advances in Virology 3   Inhibition of viral replication and reduction in cellular oxidative damage and proinflammatory mediators [37] African swine fever virus (ASFV)

Vero cell Inhibition of early and late viral protein synthesis and virion formation
Reduced viral DNA replication resulting in 98-100% reduction in viral titers [38] Human rhinovirus (HRV-16)

HeLa cell and nasal epithelia (ex vivo) Reversion of HRV-induced expression of ICAM-1
Exhibited high dose-dependent antiviral activity against HRV, leading to reduction in secretion of IL-6, IL-8, and RANTES [39] 4 Advances in Virology

Herpes Simplex Virus.
RSV was shown to inhibit the replication of herpes simplex virus-1 and herpes simplex virus-2 (HSV-1 and HSV-2) in a dose-dependent and reversible way. In this study, the authors observed a reduction in virus yield as a result of inhibition of an early event in the replication cycle: decreased production of early viral protein ICP-4. RSV also delayed interphase stage of the cell cycle and prevented virus reactivation in neuron cells that were latently infected [15]. In another study by the author using nude mice, topical application of 12.5% and 15% resveratrol ointment suppressed the development of cutaneous lesions in abraded skin infected with HSV-1 [16]. Similarly, application of 19% RSV cream on the vagina of mouse infected with HSV-2 and HSV-1 completely prevented the development of vaginal lesions, while the mortality rate was 3% as compared to the placebo group where mortality rate was 37% [14]. These remarkable effects of RSV on HSV-1 and HSV-2 infections were reported to be due to the promotion of a rapid and sustained release of ROS, which resulted in the inhibition of NF-and extracellular signal-regulated kinases/mitogenactivated protein kinases (Erk/MAPK), as well as a blockade in the expression of immediate-early, early, and late HSV genes and viral DNA synthesis [13,33].

Respiratory Syncytial Virus.
Respiratory syncytial virus (RPSV) infection is one of the most important viral diseases of the respiratory system affecting humans and it has no specific treatment. Administration of RSV in mice infected with RPSV reduced the accompanying inflammation and levels of interferon-gamma (IFN-). The mechanism here was attributed to control of toll-like receptor 3 expression, inhibition of toll/IL-1R domain-containing adaptor inducing IFN (TRIF) signaling, and induction of muscarinic 2 receptor (M2R) [9]. In an in vitro study, RSV treatment in epithelial cells inoculated with RPSV resulted in decreased production of interleukin-(IL-) 6 and a partial reduction in viral replication. There was also an inhibition of viral induced tolllike receptor domain and TANK binding kinase 1 (TBK1) protein expression [7]. RSV treatment of mice infected with RPSV was shown to increase sterile--and armadillo motifcontaining protein (SARM) expression and decrease matrix metalloproteinase 12 (MMP-12) and TIR-domain-containing adapter-inducing interferon-(TRIF) expression; these in turn decreased IFN-expression and airway inflammation and hyperresponsiveness (AHR) [6,34]. In a related study, RPSV infected mice treated with RSV also showed decreased levels of inflammatory cells and AHR. However, while RSV was able to drastically reduce the levels of nerve growth factor (NGF) after 21 days of infection, the level of brain derived neurotrophic factor (BDNF) was not significantly affected in both the treated and untreated groups [9]. Combination of RSV and baicalin (a flavonoid found in numerous species of Scutellaria) joint enema was shown to increase the levels of tumor necrosis factor-alpha (TNF-), IFN-, and IL-2 in mice infected with RPSV, which is believed to be among its antiviral mechanisms [5].

Human Immunodeficiency Virus.
In the treatment of HIV-1, a combination of RSV and decitabine (a nucleoside metabolic disorder used in the treatment of myelodysplastic syndromes) was found to be more potent than RSV alone as an anti-HIV-1 drug. However, the research also reported 15 other derivatives of RSV that were more potent as an anti-HIV-1 drug [17]. Inhibition of replication of the HIV molecular clone NL4-3 containing the mutant M184V reverse transcriptase (RT) by RSV (5 M) was reported to be associated with inhibition DNA synthesis during the reverse transcription step of the HIV life cycle. This fact was proven when administration of RSV to NL4-3 clones without the mutant M184V RT failed to inhibit viral DNA synthesis [18].  [43,44]. In DVE, viral replication was impaired by RSV via suppression of nucleic acid replication and suppression of viral capsid formation in vitro. Production of viral protein was also suppressed within the first 24 hours following infection [36]. RSV in combination with a bioflavonoid, quercetin, was shown to reduce cellular oxidative damage and secretion of proinflammatory mediators (IL-1 , IL-6, and TNF-) and chemokines (CXCL10 and CCL4), through suppression of NF-and interferon regulating factor (IRF-3), as well as viral replication in human metapneumonia (hMPV) virus infection. However, RSV did not affect viral gene transcription and protein synthesis [37]. African swine fever virus (ASFV) causes an acute hemorrhagic disease in pigs that results in up to 100% mortality. Resveratrol and oxyresveratrol (a hydroxylated analog of resveratrol) were also found to have a dosedependent effect on African swine fever virus in vitro. This was achieved through inhibition of early and late viral protein synthesis, reduced viral DNA replication, and virion formation. Hence a 98-100% reduction in viral titers was observed [38]. In human rhinovirus (HRV) infection of HeLa and nasal epithelial cells, RSV was found to exhibit a high dose-dependent antiviral activity against the virus, which was achieved through reversion of HRV-induced expression of ICAM-1. In addition, reduction in the secretion of IL-6, IL-8, and RANTES was also observed [39]. In an antiviral study of resveratrol on cytomegalovirus infection of human embryonic lung fibroblast (HEL 299), RSV prevented the synthesis of viral proteins and also inhibited virus induced activation of epidermal growth factor and phosphatidylinositol-3-kinase signal transduction. Furthermore, transcription factors of NF-and Sp1 were also inhibited. These mechanisms were observed to decrease the overall replication of the virus [40].

Hepatitis C Virus and Multiple
Sclerosis. In a study conducted by Nakamura et al. [4], RSV was found to dosedependently enhance viral RNA replication in hepatitis C virus infection in vitro. Interestingly, RSV was also reported to reverse the antiviral effects of ribavirin and interferon on HCV RNA replication and was considered nontherapeutic in the treatment of HCV infection [4]. Similarly, RSV was also found to exacerbate the clinical and histological signs of viral model of multiple sclerosis (MS), induced by Theiler's murine encephalomyelitis virus (TMEV), which belongs to the Picornaviridae [19]. Sato et al. also showed that RSV also exacerbated an autoimmune model for MS, experimental autoimmune encephalomyelitis (EAE). However, such studies are few and there are more studies highlighting the beneficial effects of RSV against viral infections, rather than its deleterious exacerbatory effects.

Conclusion
Resveratrol has shown a high antiviral potential that can be explored in both human and animal viral infections. Its main antiviral mechanisms were seen to be elicited through inhibition of viral protein synthesis, inhibition of various transcription and signaling pathways, and inhibition of viral related gene expressions. Even though there are still limitations on its bioavailability following intake, which is being widely studied, more studies should be focused on its direct use in the amelioration of viral infections in humans and companion animals.