International Journal of Infectious Diseases 24 (2014) e30–e36

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International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid

Antioxidants: potential antiviral agents for Japanese encephalitis virus infection Yu Zhang a,b, Zehua Wang a,c, Huan Chen a,b, Zongtao Chen d,*, Yanping Tian a,* a

Department of Histology and Embryology, School of Basic Medicine, Third Military Medical University, Chongqing 400038, PR China Squadron 13 of Cadet Brigade, College of Medical Laboratory Technology, Third Military Medical University, Chongqing, PR China c Squadron 17, College of Preventive Medicine, Third Military Medical University, Chongqing, PR China d Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China b

A R T I C L E I N F O

Article history: Received 18 November 2013 Received in revised form 10 February 2014 Accepted 11 February 2014 Corresponding Editor: Eskild Petersen, Aarhus, Denmark Keywords: Japanese encephalitis virus Oxidative stress Antioxidant Antivirus agents

S U M M A R Y

Japanese encephalitis (JE) is prevalent throughout eastern and southern Asia and the Pacific Rim. It is caused by the JE virus (JEV), which belongs to the family Flaviviridae. Despite the importance of JE, little is known about its pathogenesis. The role of oxidative stress in the pathogenesis of viral infections has led to increased interest in its role in JEV infections. This review focuses mainly on the role of oxidative stress in the pathogenesis of JEV infection and the antiviral effect of antioxidant agents in inhibiting JEV production. First, this review summarizes the pathogenesis of JE. The pathological changes include neuronal death, astrocyte activation, and microglial proliferation. Second, the relationship between oxidative stress and JEV infection is explored. JEV infection induces the generation of oxidants and exhausts the supply of antioxidants, which activates specific signaling pathways. Finally, the therapeutic efficacy of a variety of antioxidants as antiviral agents, including minocycline, arctigenin, fenofibrate, and curcumin, was studied. In conclusion, antioxidants are likely to be developed into antiviral agents for the treatment of JE. ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/3.0/).

1. Introduction Japanese encephalitis (JE) is prevalent throughout eastern and southern Asia and the Pacific Rim, with an estimated 50 000 cases and 15 000 deaths annually.1 In humans, JE can range from a mild febrile illness to severe encephalitis, including seizures, a polio-like illness, and a variety of movement disorders.2 In fatal cases of JE, pathological changes are seen in various parts of the nervous system, including a severe degree of vascular congestion, cerebral edema, neuron death, astrocyte activation, and microglial proliferation.1 The etiologic agent, Japanese encephalitis virus (JEV), belongs to the family Flaviviridae and can be transmitted between

* Corresponding author. Tel./Fax: +86 23 68752229. E-mail addresses: [email protected] (Z. Chen), [email protected] (Y. Tian).

animal and human hosts by Culex species of mosquitoes. Although vaccination is the most viable option to control JE, affordable vaccines are still not widely available.3 Little is known about the pathogenesis of human JEV infection, including the mechanism of its spread to the central nervous system (CNS) and viral tropism within the brain.4 JEV is a single-stranded, positive-sense RNA virus that encodes three structural proteins (capsid protein (C), precursor to the membrane protein (PrM), and envelope protein (E)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). JEV might have a peripheral replication cycle, since in vitro studies have revealed that peripheral blood mononuclear cells (PBMCs), including monocytes and macrophages, can be infected and invade the CNS via the antipodal transport of virions or through vascular endothelial cells. Neuronal apoptosis is one of the hallmarks of neurodegenerative infections and many flaviviruses

http://dx.doi.org/10.1016/j.ijid.2014.02.011 1201-9712/ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Y. Zhang et al. / International Journal of Infectious Diseases 24 (2014) e30–e36

have been shown to induce neuronal apoptosis in neurons in vitro and in rodent models in vivo.5,6 JEV NS2B–NS3 protease was found to reduce the reduction of mitochondrial membrane potential and stimulate the release of mitochondrial cytochrome C, which induces mitochondria-mediated apoptosis by activating the ASK1p38 mitogen-activated protein kinase (MAPK) signaling pathwaymediated apoptosis.7 JEV has also been shown to cause neuronal loss due to the rough endoplasmic reticulum stress pathway.8,9 Viral tropism in neural progenitor stem cells (NPSCs) and immature neurons has also been reported in experimental models of JEV infection.10–12 However, mature neurons become resistant to JEV-induced apoptosis due to the increased neuronal expression of cellular inhibitors of apoptosis, such as Bcl-2 and Bcl-x.13 In addition to neurons, astrocytes and microglial cells can also be infected by JEV.14,74 Astrocytes form part of the blood–brain barrier (BBB) and play multiple roles in the CNS, including maintaining homeostasis by storing energy in the form of glycogen and producing enzymes that exert detoxification effects. In a recent comparative study of human and mouse models, prominent astrocyte activation, particularly in areas of neuronal damage, was seen with JEV infection.15 Microglial cells are the resident macrophages of the CNS and as such might serve as a reservoir for the virus.16 Ghoshal et al. reported that the levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (Cox-2), interleukin 6 (IL-6), IL-1b, tumor necrosis factor alpha (TNF-a), and monocyte chemoattractant protein 1 (MCP-1), were significantly elevated in microglial cells following JEV infection.17 Activation of microglial cells might play a significant role in inducing neuronal cell death by stimulating the production of proinflammatory mediators. The growing number of reviews describing a role for oxidative stress in the pathogenesis of viral infections has led to increased interest in the role of oxidative stress in JEV infections.18 Reactive oxygen species (ROS)-mediated neuronal cell death, including neurons and glial cells, has been observed in vitro after JEV infection.19 This is highly specific to neuronal cells and involves an unidentified receptor-mediated death-signaling pathway. In order to avoid ROS-mediated damage caused by virus infection, antioxidants in the host cell react with elevated oxidants and superoxides and inhibit virus production. Such agents could not only alleviate disease symptoms but also decrease the long-term effects of chronic oxidative stress.20,21 This review focuses mainly

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on the role of oxidative stress in the pathogenesis of JEV infection and the potential of antioxidants for the treatment of JEV infection. 2. Oxidative stress and JEV infection Intracellular redox balance is the result of a dynamic equilibrium between oxidant and antioxidant molecules. Oxidative stress occurs when the production of ROS exceeds the capacity of cellular antioxidant defenses to remove these toxic species. Superfluous ROS are capable of causing oxidative damage to macromolecules, leading to lipid peroxidation and the oxidation of amino acid side chains and polypeptide backbones, resulting in protein fragmentation and DNA damage.22–24 Growing evidence has suggested a close correlation between oxidative stress and viral infectious disease.25 The elevated oxidants induced by viral infection include nitric oxide radicals (NO), superoxide anions (O2), hydroxy radicals (OH) and their by-products (such as hydrogen peroxide, H2O2), which may all contribute to viral pathogenesis, the modulation of cellular responses, and the regulation of viral replication and the host defense.26,27 JEV infection can lead to death in approximately 2030% of infected patients.28,29 After a mosquito bite, the virus crosses the BBB to the CNS; the resulting neuronal apoptosis and inflammation are generally attributed to JEV-induced cytopathology.30 However, the extent of cell injury that can be accredited to viral cytopathology remains unclear.17,31 Early in 2002, Liao et al. found that JEV infection induced the generation of superoxide anions (O2) in rat cortical glial cells.32 Consistent with this, a subsequent study by Srivastava et al. revealed that free radicals, such as ROS and peroxynitrite (OONO), were increased in acute JE rat models.18 After JEV infection, the elevation of O2 in the host cell leads to the activation of host superoxide dismutase (SOD) in an attempt to suppress the elevated levels of superoxide.33 H2O2, the product of the reaction between O2 and SOD, can react with O2 again if it is not rapidly cleared. The OH produced by the reaction between O2 and H2O2 then leads to host cell apoptosis. Table 1 summarizes some of the key characteristics of oxidative stress in JEV-infected cells and tissues, compiled from different studies. Additional important mechanisms regarding JEV-induced apoptosis have been reported, including the initiation of endoplasmic reticulum stress, generation of ROS, and activation of nuclear factor kappa B (NF-kB).19 Furthermore, elevated ROS can react to form peroxynitrite, which triggers the loss of adenosine

Table 1 Characteristics of oxidative stress in JEV-infected cells and tissues Cell/tissue

Stimulus/condition

Phenotypea

Ref.

Human pro-monocyte cells

Infected with JEV at MOIs of 0.1 and 1 after 24, 48, and 72 h incubation Inoculated intracerebrally with 3  106 PFU JEV Infected with JEV at MOI of 5 for 24 h Inoculated intracerebrally with 3  106 PFU JEV on days 3, 6, 10, and 20 Infected with JEV at MOI of 10

# TRX

35

" MDA, Mn-SOD; # CAT, GPx, and GSH

33

" ROS " ROS and OONO on 6 DPI; NO on 10 DPI; # ROS on 20 DPI " Superoxide anion; NO; Mn-SOD

56 18

"ROS; LDH "ROS, IL-6, IL-1b, IL-8; TRX, ceruloplasmin; EAAT-1 and EAAT-2b "ROS

31 74

Corpus striatum, frontal cortex, thalamus, and midbrain of rats Neuro2a (N2a) cells Forebrain neurons of rats Rat glial cultures (85% astrocytes, 7% microglia and others) Mouse neuroblastoma N18 cells Human astroglial cell lines (U87 and SVG cell lines) N18 and human neuronal NT-2 cells

Infected with JEV at MOI of 5 Incubated with JEV at MOI of 5 for 72h Incubated with UV-inactivated JEV

32

19

JEV, Japanese encephalitis virus; MOI, multiplicity of infection; TRX, thioredoxin; PFU, plaque-forming unit; MDA, malondialdehyde; Mn-SOD, manganese superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, glutathione; ROS, reactive oxygen species; OONO, peroxynitrite; DPI, days post inoculation; NO, nitric oxide; LDH, lactate dehydrogenase; IL, interleukin; EAAT-1, astrocytic transporters GLT-1; EAAT-2, astrocytic transporters GLAST; IP-10, interferon inducible protein 10; MCP-1, monocyte chemoattractant protein-1; MIG, membrane immunoglobulin; RANTES, regulated on activation, normal T cell expressed and secreted; UV, ultraviolet. a # = downregulated, " = upregulated, with respect to normal controls. b (A) ROS, IP-10, MCP-1, MIG, RANTES, EAAT-1 and EAAT-2 levels higher in U87 than in SVG cells. (B) Levels of TRX were less in U87 than in SVG cells.

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triphosphate (ATP) and mitochondrial membrane potential, leading to the release of cytochrome c from the mitochondria and activation of caspase 3, causing neuronal apoptosis.34 Yang et al. reported the activation of caspases 3, 8, and 9 in JEV-infected human pro-monocyte HL-CZ cells, which led to caspase-dependent apoptosis.35 However, the overproduction of O2 appears to nonselectively impair the physiological functions of host cells, regardless of infection, even though it suppresses viral replication in situ. NO and its toxic metabolite peroxynitrite are recognized as important mediators of neuronal injury. NO levels are elevated in the cerebrospinal fluid of human JE patients.36 Consistent with this, Liao et al. found that JEV infection induced the generation of NO in rat cortical glial cells.32 A subsequent study then revealed that NO levels were increased in acute JE rat models.18 JEV induces the expression of iNOS, which is thought to be a key mediator in the host innate immune response. 3. Signaling mechanisms of JEV infection in oxidative stress The molecular pathogenesis of JE remains unclear, although several potential mechanisms have been reported. JEV replication in human pro-monocyte cells has been shown to induce timedependent apoptosis. In addition, it has been shown that the downregulation of thioredoxin and the upregulation of ROS is involved in the apoptosis induced by the oxidative stress response pathway.37 JEV infection causes increased intracellular ROS production and activation of ASK1-ERK/p38 MAPK signaling, both of which are associated with JEV-induced apoptosis.35 Matrix metalloproteinase 9 (MMP-9) leads to disruption of the BBB and also contributes to the neuroinflammatory responses in many neurological diseases. Tung et al. reported MMP-9 to be overexpressed in the brains of rats with neurodegenerative diseases, which was associated with a chronic inflammatory process and the above signaling pathways. In addition, they observed the activation of the p42/p44 MAPK and the c-Jun NH(2)-terminal kinases 1/ 2 (JNK1/2) pathways in response to MMP-9 expression.38,39 Finally, treating cells with H2O2 elicited an inflammatory response involving the NF-kB signaling pathway.40,41 Therefore, there are many different potential sources of ROS in JEV-infected host cells, many of which are capable of influencing, or being influenced by, NF-kB activity. JEV infection is also associated with microglial activation, resulting in the production of proinflammatory cytokines including IL-1b and IL-18, which is mediated by ROS production.42 The oxidation of proteins is widely viewed as a signature of irreversible damage, and cellular-degradation pathways operate to remove such proteins. Ubiquitination is generally involved in protein degradation pathways. Ubiquitin is a small polypeptide that covalently attaches to proteins, either singly or in the form of polymeric chains. In the reverse process, deubiquitinase enzymes (DUBs) disassemble ubiquitin chains and strip them from their substrate proteins, thus rescuing proteins from ubiquitin-dependent degradation pathways.43 Widespread inhibition of DUBs can be caused by excess ROS generation, but this can be readily reversed by an excess of a reducing agent such as dithiothreitol.41 Zhang et al. performed a SILAC-based quantitative proteomic study of JEV-infected HeLa cells and found that the JEV infection-induced host response was coordinated primarily by the immune response, the ubiquitin–proteasome system, the intracellular membrane system, and lipid metabolism-related proteins.44 Expression of interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, was found to be rapidly induced by interferon-a/b (IFN-a/b), and ISG15 conjugation to target proteins was associated with the antiviral immune response.45 Hsiao et al. reported that ISG15 overexpression significantly reduced the JEV-induced cytopathic

effect and inhibited JEV replication. Furthermore, ISG15 overexpression increased the phosphorylation of interferon regulatory factor-3 (IRF-3, Ser396), the Janus/just another kinase-2 (JAK2, Tyr1007/1008), and signal transducers and activators of the transcription 1 (STAT1, Tyr701 and Ser727) in JEV-infected cells. ISG15 overexpression also induced the translocation of the transcription factor STAT1 to the nucleus and activated the expression of STAT1-dependent genes including IRF-3, IFN-b, IL8, protein kinase R (PKR), and 20 ,50 -oligoadenylate synthetase (OAS), both before and after JEV infection.46 These data reveal that ubiquitination is involved in JEV infection, but the specific mechanism is unclear. 4. The effects of changes in antioxidants on JEV infection Reducing conditions are normally maintained within the cell by antioxidant molecules. The antioxidant system is classified into two types: an enzyme system including SOD, catalase (CAT), and glutathione peroxidase (GPx), and other molecules including glutathione (GSH), N-acetylcysteine (NAC), and selenium.47 In addition, dietary micronutrients also contribute to the antioxidant defense system including b-carotene, vitamin C, and vitamin E. Water-soluble molecules, such as vitamin C, are potent radical scavenging agents in the aqueous phase of the cytoplasm, whereas lipid soluble antioxidants, such as vitamin E and b-carotene, act as antioxidants within lipid environments.48 Selenium, copper, zinc, and manganese are also important elements, since they act as cofactors for antioxidant enzymes.49 In order to avoid harmful effects caused by oxidants, antioxidants in the host cell react with elevated oxidants and superoxides in several ways under normal conditions (Table 2). 4.1. SODs and CAT SODs are metal-containing proteins that catalyze the removal of superoxide, generating water peroxide as the final product of dismutation. Three major isoforms of SODs have been identified. The copper–zinc SOD (CuZn-SOD) isoform is present in the cytoplasm, nucleus, and plasma. In contrast, the manganese SOD (Mn-SOD) isoform is primarily located in mitochondria. Finally, iron SOD (Fe-SOD) is mainly found in the plastids of plants and prokaryotes.50 CAT is a family of heme-containing enzymes that convert H2O2 to water and O2. They are predominantly localized in peroxisomes, although expression can also be detected in the mitochondria and endoplasmic reticulum.48 Therefore, intracellular H2O2 cannot be eliminated unless it diffuses into peroxisomes. JEV infection has been reported to induce the generation of O2 and NO in rat cortical glial cells. Mn-SOD, but not CuZn-SOD, was activated by JEV infection and this activation was blocked by pyrrolidine dithiocarbamate. In addition, increased SOD activity was apparent in cell lines that had been acutely or persistently infected with JEV.32 These results suggest that cellular factors regulating oxidative pathways might play roles in the cellular response to JEV infection. In addition, Kumar et al. reported that the activity of Mn-SOD in the striatum, cortex, thalamus, and Table 2 Antioxidants in the host cell react with the elevated oxidants SOD

2O2  + 2H+ ! H2O2 + O2 CAT

2H2O2 ! 2H2O + O2 GPx

H2O2 + 2GSH ! 2H2O + GS-SGa GPx

2GSH + R-O-OH ! GS-SG + H2O + R-OHa SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase. a GS-SG, the product of these reactions, is converted to GSH (which does no damage to the host cell) under the effect of glutathione reductase.

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midbrain was increased significantly in JEV-infected rats, whereas the activity of CAT, GPx, and GSH all decreased significantly compared with controls. Although Mn-SOD removes superoxide radicals to combat oxidative stress, the simultaneous reduction of CAT and GPx levels means that the H2O2 cannot be scavenged.33 4.2. GSH and GPx The most abundant cellular antioxidant is the cysteinecontaining tripeptide GSH, which prevents the oxidation of protein thiol groups either directly by reacting with reactive species, or indirectly through glutathione transferases.35,51 GPx remove H2O2 by coupling its reduction with the oxidation of GSH. GPx can also reduce other peroxides such as fatty acid hydroperoxides. These enzymes are present in the cytoplasm at millimolar concentrations and also in the mitochondrial matrix. Early in 1997, Garaci et al. reported that HIV-1 infection induced a significant decrease in intracellular reduced GSH levels in human macrophages in vitro.52 Consistent with this, Nencioni et al. demonstrated that redox changes during influenza A infection were mediated by a depletion of GSH. They found that GSH levels decreased during viral replication only in those cell lines that efficiently produced mature influenza A virus.53 It is possible that the concentration of GSH in the host cell has a significant effect on viral replication. In 2010, we showed that infection with dengue virus serotype 2 (DENV2), also belonging to the Flaviviridae family, resulted in a decrease in intracellular GSH. In contrast, supplementing with GSH significantly inhibited the activation of NF-kB, resulting in decreased production of DENV2 in HepG2 cells.54 In an additional study, the levels of GSH were decreased significantly in all the brain regions of rats after JEV infection compared with controls.33 Therefore, growing evidence has indicated that GSH might be valuable for the prevention of JEV infection. 4.3. NAC NAC is a precursor in the formation of the antioxidant glutathione. Early in 1990, Staal et al. found that H2O2 promotes the replication of HIV, but that antioxidants such as NAC have the opposite effect.55 The group of Raung et al. reported that none of the antioxidants tested, including NAC, protected N18 cells from JEV-induced apoptotic and necrotic cell death.31 In addition, Mishra et al. revealed that NAC was ineffective at protecting cells from JEV-induced death.56 However, in both these studies, NAC was able to change the intracellular redox status by scavenging oxygen radicals. 5. Antioxidant agents inhibit JEV production Along with the important role of oxidative stress in JEV infection, a variety of antiviral agents have been studied to

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evaluate their therapeutic efficacy. Here we review four agents that modulate different pathways (Table 3). 5.1. Minocycline Minocycline, a semisynthetic tetracycline, is neuroprotective in animal models against a number of acute CNS injuries, neurodegenerative diseases, and CNS infection.57 In addition to its antioxidant properties, minocycline has been reported to have many cellular effects, such as stimulating the expression and activity of caspases and nitric oxide synthase, reactive microgliosis, and the induction of the anti-apoptotic protein Bcl-2.58–60 Mishra et al. reported that minocycline inhibits JEV-induced free radical generation in mouse neuroblastoma N2a cells. They measured lactate dehydrogenase (LDH) activity, ROS levels, and mitochondrial membrane potential and found that 20 mM minocycline inhibited ROS production and neuronal death. There was a significant increase in the expression of phospho-NF-kB, heat-shock protein 70 (HSP-70), phospho-JNK, and phospho-p38 MAPK in JEV-infected mice; however, treatment with minocycline reduced this expression significantly. The ratio of Bax/Bcl-2 was also decreased significantly by treatment with minocycline in JEVinfected N2a cells.56 These findings suggest that minocycline reduces the neuronal damage induced by JEV infection in neuronal cell culture models, in part by inhibiting oxidative stress. 5.2. Arctigenin Arctigenin is a plant lignan with antioxidant, anti-inflammatory, and antiviral activities that modulates multiple cellular machineries.61–63 It has been described as a biologically active lignan, with potent anti-HIV and anti-influenza virus effects.64–66 Arctigenin has been found to confer complete protection to animals infected with JEV and significantly reduce morbidity. Treatment with arctigenin increased the levels of SOD1, but significantly reduced the levels of NO and iNOS in the JEV-infected mouse brains; this abrogated microglial activation and the induction of proinflammatory cytokines. JEV infection upregulated the induction of several stress-associated proteins, and treatment with arctigenin significantly reduced the level of these proteins in infected animals. In particular, the expression of phospho-p38 MAPK, phospho-c-Jun, HSP-70, phospho-ERK1/2, and phospho-Akt were decreased. In vitro, arctigenin treatment was found to reduce viral titers and decrease the intracellular viral load in JEV-infected N2a cells. In vivo and in vitro experiments clearly revealed that arctigenin reduced viral load and replication, as well as inhibiting the neuronal death, secondary inflammation, and oxidative stress that resulted from microglial activation.67 Therefore, the antiviral, neuroprotective, anti-inflammatory, and antioxidant effects of arctigenin successfully reduced the severity of JEV-induced disease.

Table 3 Recently discovered antioxidative agents for JEV infection Agent

Target

Active concentration

Model system

Ref.

Minocycline Arctigenin

Inhibition of oxidative stress Oxidative stress resulting from microglial activation A peroxisome proliferator-activated receptor alpha (PPARa) agonist

20 mM Twice daily (10 mg/kg body weight) for the next 7 days Mice: 100 mg/kg 4 days prior to JEV exposure Cell: 40 mg/ml 1 h prior to exposure to JEV Treated with 1, 5, and 10 mM doses of curcumin for 8 h

Mouse neuroblastoma (N2a cells) BALB/c mice

56 67

BV-2 microglial cell line and BALB/c mouse models

71

Neuro2a cell line

73

Fenofibrate

Curcumin

Decreasing the cellular reactive oxygen species level

JEV, Japanese encephalitis virus.

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5.3. Fenofibrate Fenofibrate is an agonist of peroxisome proliferator-activated receptor a (PPARa) and so it stimulates the expression of the neuroprotective genes that trigger both antioxidant and antiinflammatory responses. It increases the transcription of a number of genes including CuZn-SOD, GPx, glutathione reductase (GR), glutathione-S transferase (GST), and CAT, but inhibits the transcription of NF-kB, STATs, activator protein 1 (AP1), and nuclear factor of activated T cells (NFAT) signaling pathways.68,69 PPARa activation by fenofibrate confers neuroprotection in various CNS disorders, such as traumatic brain injury and Parkinson’s disease.69,70 Sehgal et al. examined the neuroprotective effects of fenofibrate using both in vivo and in vitro models of JEV infection. In JEV-infected BALB/c mice, pretreatment with fenofibrate for 4 but not 2 days reduced mortality by 80%. In addition, brain leukotriene B4 (LTB4) levels decreased concomitantly with the induction of Cyp4f15 and 4f18, which catalyze the detoxification of LTB4 via hydroxylation. They also found that fenofibrate abrogated JEV-mediated microglial activation and the release of inflammatory mediators in the BV-2 microglial cell line. Pretreatment with fenofibrate for 4 days significantly decreased the viral titers in the culture medium of BV-2 cells infected with JEV.71 Thus fenofibrate, a PPARa agonist that is commonly used as a hypolipidemic drug, could potentially be used for treatment or as prophylaxis during JE epidemics to reduce mortality and morbidity. 5.4. Curcumin Curcumin is a naturally occurring phenolic compound extracted from the rhizome of Curcuma longa L.72 It has been reported to have anti-inflammatory, antioxidant, and anti-prolif-

erative properties and modulates multiple cellular machineries by inhibiting several intracellular signaling pathways including MAPK, PI3K/AKT, and pNF-kB.63 JEV-infected N2a cells were treated with varying doses of curcumin in vitro. Cell viability was increased and apoptosis was decreased significantly in curcumin-treated cells compared with cells infected with only virus. Curcumin increased expression of SOD-1 and HSP-70, but decreased cellular ROS levels; this helped ameliorate the oxidative stress and apoptosis caused by a variety of factors. Curcumin restored the integrity of cellular membranes by decreasing the expression of pro-apoptotic signaling molecules and modulating the cellular levels of stress-related proteins such as pJNK, phospho-p38 MAPK, pERK-1/2, and pNF-kB. Curcumin also reduced the production of infectious viral particles from previously infected neuroblastoma cells by dysregulating the ubiquitin–proteasome system.73 These studies revealed that curcumin has an outstanding safety profile and substantial potential antiviral effects. 6. Conclusions and perspectives Unfortunately, no specific antiviral agents are currently approved for the treatment of JEV infection in human patients. An increasing number of drug-resistant viruses have emerged due to the abuse of traditional antiviral drugs. Great efforts are being made in laboratories worldwide to identify an effective, safe, and readily available therapy to alleviate JEV. In this review, we have summarized the relationship between oxidative stress, JEV infection, and a variety of antioxidants as antiviral agents to evaluate their therapeutic efficacy (Figure 1). Antiviral therapies based on antioxidants are promising potential targets and it is likely that more antioxidants will

Figure 1. Mechanistic basis and multi-level regulation of antioxidant-related pathways in Japanese encephalitis virus (JEV) infection. (A) JEV infection causes upregulation of ROS and downregulation of antioxidant, which is involved in the apoptosis induced by the oxidative stress response pathway. JEV infection activates p-NF-kB, p-JNK, p-p38 MAPK, and DUBs signaling, which is associated with JEV-induced apoptosis. (B) Treatment with antioxidants, such as minocycline, arctigenin, fenofibrate, and curcumin, increases the transcription of a number of genes including SOD, GPx, GR, HSP-70, GST, and CAT, but inhibits the transcription of NF-kB, STATs, AP1, and NFAT signaling pathways. Antioxidants reduce the viral load and replication, as well as inhibiting the neuronal death, secondary inflammation, and oxidative stress resulting from microglial activation. Abbreviations: AP1, activator protein 1; CAT, catalase; Cyt c, cytochrome c; DUBs, deubiquitinase enzymes; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S transferase; HSP-70, heat-shock protein 70; NF-kB, nuclear factor kappa B; ROS, reactive oxygen species; STATs, signal transducers and activators of transcription; NFAT, nuclear factor of activated T cells; SOD, superoxide dismutase.

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be identified with potential as therapeutic agents against JEV infection. With many potential candidates based on antioxidants and the support of government and private funding, the prospect of an effective antiviral or therapeutic compound for the treatment of JEV is encouraging. Funding source: This work was supported by grants from the National Natural Science Foundation of China (30901372 and 31200898) and the Science Foundation of the Third Military Medical University to a faculty (2011XQN02). Conflict of interest: All authors declare no competing interests.

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