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Review

1.

Introduction

2.

JEV and its diseases

3.

Antiviral drugs

4.

Conclusion

5.

Expert opinion

Potential chemotherapeutic targets for Japanese encephalitis: current status of antiviral drug development and future challenges Tomohiro Ishikawa & Eiji Konishi† †

Osaka University, Research Institute for Microbial Diseases, BIKEN Endowed Department of Dengue Vaccine Development, Osaka, Japan

Introduction: Japanese encephalitis (JE) remains a public health threat in Asia. Although several vaccines have been licensed, ~ 67,900 cases of the disease are estimated to occur annually, probably because the vaccine coverage is low. Therefore, effective antiviral drugs are required to control JE. However, no licensed anti-JE drugs are available, despite extensive efforts to develop them. Areas covered: We provide a general overview of JE and JE virus, including its transmission cycle, distribution, structure, replication machinery, immune evasion mechanisms and vaccines. The current situation in antiviral drug development is then reviewed and future perspectives are discussed. Expert opinion: Although the development of effective anti-JE drugs is an urgent issue, only supportive care is currently available. Recent progress in our understanding of the viral replication machinery and immune evasion strategies has identified new targets for anti-JE drug development. To date, most candidate drugs have only been evaluated in single-drug formulations, and efficient drug delivery to the CNS has virtually not been considered. However, an effective anti-JE treatment is expected to be achieved with multiple-drug formulations and a targeted drug delivery system in the near future. Keywords: antiviral, broad spectrum, central nervous system, in silico modeling, Japanese encephalitis, nucleic acid-based, virus replication cycle-based Expert Opin. Ther. Targets [Early Online]

1.

Introduction

Japanese encephalitis virus (JEV), a member of the genus Flavivirus in the family Flaviviridae, is distributed throughout east, southeast, and south Asia [1]. Japanese encephalitis (JE), caused by JEV infection transmitted through infected mosquito bites, is a major viral encephalitis in these areas, and therefore a major public health problem. Because JEV is maintained in nature in birds and vector mosquitoes, independently of humans, its elimination is difficult. Various vaccines have been developed to prevent JE, and the introduction of these vaccines into routine vaccination programs has successfully reduced the numbers of JE cases in several countries, indicating that JE is a vaccine-preventable disease [2]. Currently, Verocell-derived inactivated vaccines (IXIARO/JESPECT, manufactured by Valneva SE; JEEV by Biological E; JEBIKV by BIKEN; ENCEVAC by Kaketsuken; JENVAC by Bharat Biotech; JEVAC by Liaoning Chengda Biotechnology Co.), a live-attenuated vaccine (SA14-14-2/CD.JE-VAX, manufactured by Chengdu 10.1517/14728222.2015.1065817 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

1

T. Ishikawa & E. Konishi

Article highlights. . . . . .

Japanese encephalitis (JE) virus infection remains a major public health concern in endemic regions. Several preventive vaccines are currently available worldwide. No specific anti-JE drugs have been licensed. The combination of several compounds is considered a key therapeutic strategy. Efficient drug delivery measures are required, particularly to infected cells in the CNS.

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This box summarizes key points contained in the article.

Institute of Biological Products) and a chimeric vaccine (IMOJEV/THAIJEV manufactured by Sanofi Pasteur and GPO-MBP) are licensed throughout the world [2]. Despite the availability of these vaccines, it is estimated that ~ 67,900 cases, including 20,400 deaths, occur worldwide every year [3], probably because the vaccine coverage is low, particularly in developing countries. Therefore, effective antiviral drugs are required to combat JEV infection. In contrast to vaccine development, no approved antiviral drugs are currently available for the treatment of JE, so only supportive care can be given. Together with efforts to increase vaccine coverage, the development of effective therapeutic antiviral drugs to control JE is an urgent issue. In this review, following general overviews of JE and JEV, the current status and future perspectives on the development of anti-JE drugs are described. 2.

JEV and its diseases

2.1

Transmission cycle of JEV

Virion structure and functions of the viral proteins

2.3

JEV was initially isolated from the human brain in 1935, and then isolated from Culex tritaeniorhynchus mosquitoes in 1938, both in Japan [2]. In nature, JEV is maintained in a bird--mosquito transmission cycle. Although > 90 species of birds are susceptible to JEV, egrets and herons are known to display high viremic titers [4]. Therefore, wild birds are considered to act as the reservoirs of the virus, and perhaps as the amplifying hosts [5,6]. Antibodies against JEV have been found in many mammals, including dogs, cattle and wild boars [5,7,8]. Among these, swine can develop a durable hightiter viremia (up to 4 days) and only swine are considered to be important amplifying hosts [5]. C. tritaeniorhynchus mosquitoes are recognized as the principal vector for human infection, although JEV has been isolated from > 30 species of mosquitoes, including in the genera Culex, Aedes and Anopheles [9]. Humans and horses develop the disease, but neither shows a durable viremic titer high enough for them to transmit the virus to mosquitoes. Therefore, humans and horses are recognized as dead-end hosts [2]. Global distribution Since the first isolation of JEV in 1935, its circulation has been confirmed in eastern, southern and southeastern Asian 2.2

2

countries (Figure 1). In Pakistan, the JEV genome was first detected in cerebrospinal fluid specimens in 1992 [10], although suspected JE cases had been reported before then [11]. In Australia, JEV was first isolated from human sera in the Torres Strait Islands, located north of the Australian mainland, in 1995 [12]. Following the continuous activity of JEV in this region, the JEV genome was finally isolated from mosquitoes collected from the Cape York Peninsula on the Australian mainland [13]. Therefore, the distribution area of JEV is still expanding eastward and southward. JEV is divided into five distinct genotypes [14], and all five are only found together in the Indonesia--Malaysia region [14]. Genotype II is mainly distributed in the Australia--New Guinea region, whereas genotype III is widely distributed, particularly in temperate regions [14]. Neither genotype IV nor V had been found outside the Indonesia--Malaysia region until 2009 (see below). In the past few decades, the dominant genotypes in many countries have been displaced (Figure 1) [2], although the details of the mechanisms underlying these changes remain unclear. The replacement of genotype III with genotype I has been observed in China, India, Japan, South Korea, Taiwan, Thailand and Vietnam, and the replacement of genotype II with genotype I has been observed in Australia [2]. In addition to these phenomena, genotype V, which was previously only associated with the Indonesia--Malaysia region, suddenly emerged in China and South Korea in 2009 and 2010, respectively [15,16].

Approximately 11-kb positive-strand RNA genome of JEV encodes three structural proteins (capsid [C]; precursor membrane [prM]; envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) in a single open reading frame flanked by 5¢- and 3¢-untranslated regions (UTRs) (Figure 2) [17]. The single large synthesized polyprotein is cleaved into the 10 viral proteins by host and viral proteases. The envelope of the immature virion particle contains 180 copies of the prM--E heterodimer and three prM--E heterodimers form one spike on the virion surface [18]. During the maturation steps, the prM protein is cleaved to form the matured M protein by the trans-Golgi enzyme furin [19], forming 90 copies of the E--E homodimer on the mature virion particle [20]. The Flavivirus nucleocapsid consists of homodimers of C protein complexed with genomic RNA [21]. The C protein is reported to form a homodimer [22] that is important for viral replication and nucleocapsid formation [23,24]. The prM protein acts as a chaperone for the proper folding of the E protein [25] and is believed to prevent the low-pH-triggered oligomeric rearrangement of E proteins by covering them (see below) [17]. The E protein, a class II fusion protein, plays roles in receptor binding and

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Potential chemotherapeutic targets for JE

Figure 1. Illustration showing the distribution of JEV and genotype replacements. Areas of the worldwide distribution of JEV are shown in gray. Replacement of the dominant genotype and the estimated year when the replacement occurred are indicated in boxes. JEV: Japanese encephalitis virus.

5′UTR

3′UTR 5′CS C prM

E

NS1

NS2A NS2B

NS3

Complexed with NS3 Complexed with RNA viral replication

NS4A NS4B

NS5

RNA replication?

3′CS

RNA replication?

Chaperon for E Receptor binding Membrane fusion

RNA synthesis assembly blocking PKR

RNA replication? blocking IFN signaling

Serine protease RNA helicase Nucleoside triphosphatase

Guanylyltransferase Methyltransferase RNA dependent RNA polymerase Blocking IFN signaling

Figure 2. Illustration showing the JEV genome organization and functions of the viral proteins. The JEV genome structure and the encoded JEV proteins are shown. The major functions of each viral protein are indicated below. C: Capsid; E: Envelope; JEV: Japanese encephalitis virus; NS: Nonstructural; PKR: Protein kinase R; prM: Precursor membrane; UTR: Untranslated region.

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T. Ishikawa & E. Konishi

membrane fusion [17] and is also known as a highly immunogenic protein, containing most of the neutralizing viral epitopes [26]. The Flavivirus NS1 protein exists in an intracellular dimeric form and an extracellular hexameric form [27]. Although it is suggested to be involved in RNA replication because it co-localizes with double-stranded RNA (dsRNA) [28], the function of the JEV NS1 protein is unclear. The Flavivirus NS2A protein is known to be important for viral RNA synthesis and virion assembly [29], but the function of the JEV NS2A protein is poorly understood. The NS2B protein, a small membrane-associated protein, complexes with the NS3 protein, and in the N-terminal one-third of the NS3 protein has a serine protease activity when complexed with NS2B [30]. The remaining C-terminal region of NS3 has RNA helicase and nucleoside triphosphatase domains [31,32]. During the replication cycle, NS3 interacts with NS5 [33]. Although the Flavivirus NS4A protein, a small hydrophobic protein, is implicated in RNA replication by coordinating with NS1 [34], its functions essentially remain unclear. Similarly, the functions of the Flavivirus NS4B protein are hardly known, although it is suggested to be involved in RNA replication because it co-localizes with dsRNA [35]. The NS5 protein is a multi-enzymatic protein, like NS3 protein. The N-terminal region of NS5 has a guanylyltransferase/methyltransferase domain, which participates in capping the 5¢ terminus of the genomic RNA [36], and the C-terminal region has an RNA-dependent RNA polymerase domain [37]. Viral replication in infected cells The JEV replication machinery in infected cells is shown in Figure 3. To date, several candidate JEV entry receptors or attachment factors have been identified. Because JEV has a wide host range, including both vertebrate and invertebrate animals, and because JEV infects several types of cells/tissues before invading the CNS of the dead-end host, it must use multiple receptors. Heparan sulfate [38], heat shock protein 70 [39], integrin [40], CLEC5A [41], vimentin [42] and CD4 [43] have been reported to be JEV receptors on mammalian cells. The receptor-bound virion is internalized by a clathrin-dependent [44] or clathrin-independent [45] pathway, probably according to the cell type. The low pH inside the endosome triggers the oligomeric rearrangement of the virion surface E--E homodimers to form E--E--E homotrimers, exposing a fusion loop [46]. Following membrane fusion, the genomic RNA is released into the cytoplasm. Several characteristic membrane structures are induced in Flavivirusinfected cells [47]. RNA replication occurs inside the membrane structures, confirmed by dsRNA localization, and the translation of viral proteins occurs in an adjacent compartment [48,49]. During the viral replication process, the Flavivirus genomic RNA is cyclized via two complementary sequences located at the 5¢ and 3¢ termini of the genome [50]. This cyclization of the genome is thought to facilitate 2.4

4

the efficient recruitment of ribosomes. Several host factors have been implicated in efficient viral replication. Mov34, La, polypyrimidine tract-binding protein, far upstream element-binding protein 1, glyceraldehyde-3-phosphate dehydrogenase, heterogeneous nuclear ribonucleoprotein A2, DEAD-box RNA helicase DDX5 and DDX3 have been shown to interact with the JEV genome, particularly with the UTRs [51-58]. The immature virion is believed to bud into the lumen of the endoplasmic reticulum, to be transported via the host secretory pathway, and is then released from the cell by exocytosis [59]. As described above, the prM protein on the virion surface is cleaved to M protein by the trans-Golgi enzyme furin before exocytosis [19]. Immune evasion strategy On infection, many microbes take measures to evade the host immune responses. Several mechanisms utilized by JEV have been identified (Figure 3). IFNs produced by infected cells are known to be important modulators of the cellular antiviral responses [60]. In JEV-infected cells, the cytosolic dsRNA is sensed by retinoic acid-inducible gene-I, leading to IFN production [61]. To evade this sensor, the viral dsRNA is thought to be concealed within an intracellular membrane in porcine cells, delaying its exposure to the host immune response [62]. The signals transduced when IFN binds to the IFN receptors regulate the expression of numerous IFN-stimulated genes via the JAK/STAT (Janus kinase and signal transducer and activator of transcription) pathway to establish an antiviral state in the cells [60]. The JEV NS5 protein inhibits the phosphorylation of TYK2 and STAT1 [63] and NS4A is also reported to partially block the phosphorylation of STAT1 and STAT2 [64]. The JEV NS2A protein blocks the activation of protein kinase R (PKR), which is an IFN-induced antiviral response [65]. Although the responsible elements have not been identified, antiviral viperin, one of the IFN-stimulated genes, is degraded via the proteasome-mediated pathway in JEV-infected cells [66]. The accumulation of short fragmented RNA derived from the 3¢-UTR, a region highly conserved among the flaviviruses, is observed in infected cells [67]. These short fragmented RNAs inhibit IFN-b production by blocking the phosphorylation of IFN regulatory factor-3 [68]. Although autophagy is induced to maintain cellular homeostasis, several viruses utilize autophagy for their efficient replication [69]. Recently, JEV was demonstrated to induce autophagy for its efficient replication, resulting in reduced IFN production [70]. In addition to these strategies for evading the innate immune responses of the host, JEV uses another measure against the acquired immune responses. On JEV infection, particularly of dendritic cells and macrophages, MHC class I-restricted antigen presentation is inhibited [71], so poor CD8+ T-cell responses are induced [71,72]. Together with the inhibition of the cellular immune responses, a regulatory T-cell population that suppresses the immune responses is expanded by JEV infection [73]. 2.5

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Potential chemotherapeutic targets for JE

Immune evasion

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Virus replication

Nucleus

Golgi ER

Figure 3. Illustration showing the JEV replication and immune evasion machinery. The JEV replication machinery in infected cells is shown on the right side of the figure, with boxes indicating the major steps. Boxes also indicate potential antiviral drug targets. The major immunological cascades stimulated against JEV infection are represented on the left. Boxes indicate the immune evasion strategies used by JEV, which are also potential antiviral drug targets. 2¢,5¢-OAS: 2¢-5¢-Oligoadenylate synthetase; ISRE: IFN-stimulated response elements; JEV: Japanese encephalitis virus.

Diseases Human JEV infections usually result in asymptomatic infections, with symptomatic-to-asymptomatic infection ratios ranging from 1:25 to 1:1000 [74]. The ratios reported in non-endemic areas are often higher than those in endemic areas. The disease is normally initiated with nonspecific febrile-illness-like manifestations, including high fever, headache, diarrhea and vomiting, following a 5- to 15-day incubation period. In severe cases, patients then develop seizure, polio-like flaccid paralysis, meningitis and encephalitis. Hyperventilation and extrapyramidal features are strongly associated with JEV infection and are therefore diagnostic symptoms [75]. The typical case fatality is 20 -- 30%, although this depends in large part on the medical facilities available [76]. Up to 50% of survivors suffer long-term psychoneurological sequelae. 2.6

Vaccines In contrast to the limited therapeutic measures available, several effective vaccines have been developed and are widely 2.7

used. The detailed history of vaccine development and the current situation have already been reviewed [2]. The first vaccine, a mouse-brain-derived inactivated vaccine based on the JEV Nakayama strain, was developed in 1954 in Japan, and the vaccine strain was changed to Beijing-1 for domestic use in 1989 [2]. Although the mouse-brain-derived inactivated vaccine has been available worldwide as the routine vaccine and the traveler’s vaccine, World Health Organization (WHO) position papers recommended in 2006 and 2015 that the mouse-brain-derived inactivated vaccine should be replaced with a new-generation vaccine [77], and in fact Vero-cellderived inactivated vaccines based on the SA14-14-2 and Beijing-1 strains (since 2009) and the 821564 XZ strain (since 2013) have been available in many countries (Table 1) [2]. This replacement was prompted by the recognition of substances potentially derived from mouse brain that can cause severe adverse effects, such as acute disseminated encephalomyelitis, and ethical issues about the use of numerous animals to prepare the vaccine antigens. Vero-cell-derived inactivated vaccines are currently licensed in > 10 countries, including

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T. Ishikawa & E. Konishi

Table 1. Current Japanese encephalitis vaccines. Vaccine type

Antigen

Availability

Prequalification by WHO

Vero cell-derived inactivated vaccine

SA14-14-2

Europe, US, Canada, Hong Kong, Singapore, Israel, Taiwan (expected in 2017) For pediatric use, US, European Union, Norway, Liechtenstein, Iceland Australia, New Zealand, India Japan Japan India China China, Cambodia, India, Laos, Myanmar, Nepal, North Korea, South Korea, Sri Lanka, Thailand Australia, Brunei, Malaysia, Myanmar, the Philippines Thailand

No

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Live-attenuated vaccine Chimeric vaccine

SA14-142 Beijing-1 Beijing-1 821564 XZ Beijing-P3 SA14-14-2 SA14-14-2 SA14-14-2

Yes (July 2013) No No No No Yes (October 2013) No Yes (September 2014)

WHO: World Health Organization.

non-endemic countries, and the vaccine produced by Biological E in India was the first vaccine against JE, which was prequalified by WHO in July 2013 [2]. A live-attenuated vaccine based on the SA14-14-2 strain has been used in China since 1988 (Table 1) [2]. In China, this live-attenuated vaccine and a Vero-cell-derived inactivated vaccine based on the BeijingP3 strain, which has been available since 2008, are displacing the primary hamster kidney-cell-derived inactivated vaccine that had also been used in China (Table 1) [2]. Currently, the SA14-14-2-based live-attenuated vaccine is approved in 10 Asian countries (Table 1) [2]. In October 2013, the liveattenuated vaccine produced by the Chengdu Institute of Biological Products in China was prequalified by WHO as the first vaccine for pediatric use against JE [2]. A liveattenuated chimeric vaccine based on the yellow fever virus vaccine strain 17D was developed containing the prM--E genes of strain SA14-14-2 in place of the corresponding genes of strain 17D [2]. It has been licensed in Australia and Thailand since 2010 and is currently also available in Brunei, Malaysia, Myanmar and the Philippines. This chimeric vaccine, produced by GPO-MBP in Thailand, was prequalified by WHO in September 2014. JEV activities have been controlled well in several countries by the introduction of these vaccines into routine immunization programs. The improved vaccine was introduced into the routine immunization program in Japan in 1967, when > 1000 cases per year occurred, and the number of reported cases has since decreased markedly, with < 10 cases reported annually since 1992 [78]. Similar trends have been observed in China [79], Korea [80,81], Malaysia [82], Nepal [83], Sri Lanka [84], Taiwan [85], Thailand [86,87] and Vietnam [88]. These observations clearly indicate that JE is a vaccinepreventable infectious disease. However, it is estimated that ~ 67,900 cases, including 20,400 deaths, occur worldwide annually [3], probably because the vaccine coverage is low. Consistent with this, countries that have successfully 6

controlled JEV activities, as described above, have high vaccine coverage (Table 2). To increase the vaccine coverage in endemic countries, the GAVI Alliance (formerly known as the Global Alliance for Vaccines and Immunization) recently offered financial support for immunization campaigns and the subsequent introduction of JE vaccines into routine immunization programs in these countries [89]. 3.

Antiviral drugs

Because JE is a vaccine-preventable mosquito-borne infectious disease, the immunization of all people at risk and the control of the mosquito vector are considered rational measures to combat JEV. However, the most recent estimate showed that ~ 67,900 cases of JE occur annually throughout the world, despite the availability of licensed vaccines [3]. Therefore, in the current situation, effective antiviral drugs are required. In contrast to the successful vaccine development, there are no approved anti-JE drugs, regardless of the extensive efforts made to develop them. Therefore, only supportive care has been available (refer to the guideline [90]). Here, we mainly review the candidate anti-JE drugs evaluated in vivo (Table 3), and then discuss the potential targets of anti-JE drug development. Nonspecific broad-spectrum antiviral drugs Among the well-known broad-spectrum antiviral drugs, IFN has been studied extensively and approved for clinical use, including against infection by hepatitis C virus, another member of the family Flaviviridae. Therefore, IFN was expected to be effective against JEV infections, and in fact, displayed antiJEV effects in vitro [91]. However, a randomized, double-blind human clinical trial conducted in Vietnam showed that daily intramuscular injections of IFN-2a for 7 days did not improve the outcomes of JE patients [92]. Ribavirin (1-b-d-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) is also a broad-spectrum 3.1

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Potential chemotherapeutic targets for JE

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Table 2. Current status of immunization programs and vaccine coverage in Asian countries. Country

Immunization program

Implementation year

Vaccine coverage (%)*

Australia Bangladesh Bhutan Cambodia China India Japan Laos Malaysia Myanmar Nepal Papua New Guinea Philippines Singapore South Korea Sri Lanka Taiwan Thailand Vietnam

Risk areas only None None Subnational National Risk areas only National None Subnational None Subnational None None None National National All areas National Subnational

NA NA NA 2009 2008† NA 1967z NA 2001 NA 2006 NA NA NA 1971 1988 1968 1985 1997

NA NA NA NA 99 69 99 NA 98 NA 72 NA NA NA 99 99 NA 92 91

*Estimated vaccine coverage in 2013 [171]. † Although immunization programs were implemented before 2008 in several provinces, the nationwide program was initiated in 2008. z The nationwide program using the improved vaccine has been implemented since 1967. NA: Not applicable.

Table 3. Anti-Japanese encephalitis drugs reviewed. Category

Antiviral drugs

Target/mechanism

Ref.

Nonspecific broad spectrum

IFN Ribavirin Minocycline Arctigenin Fenofibrate Curcumin Pentoxifylline Nitazoxanide siRNA PNA Morpholino oligomer Heparan sulfate E-binding peptide NSQ Indirubin Bovine lactoferrin Griffithsin Recombinant E MCPIP1 Kaempferol SCH16 Ivermectin 4-hydroxypanduratin

ISG Inosine monophosphate dehydrogenase, etc. Antioxidant Antioxidant Antioxidant Antioxidant Assembly or Release Early/mid-replication cycle C, M, E, NS1, NS3, NS4B, NS5 UTR UTR Receptor binding Receptor binding Attachment Attachment Receptor binding Receptor binding Receptor binding RNA replication RNA replication Translation NS3 NS2B/NS3

[91,92] [94,95] [99,100] [101] [102] [103] [105] [107] [109-115] [119] [121,122] [123-125] [126] [127] [128] [129] [130] [131,132] [133] [134] [138,139] [146] [147]

Nucleic acid-based

Virus replication cycle-based

In silico modeling-based

C: Capsid; E: Envelope; ISG: IFN-stimulated gene; M: Membrane; MCPIP1: Monocyte chemoattractant protein 1-induced protein 1; NS: Nonstructural; NSQ: Nanoscale silicate platelet; PNA: Peptide nucleic acid; siRNA: Small-interfering RNA; UTR: Untranslated region.

antiviral drug, which acts by inhibiting inosine monophosphate dehydrogenase, exerting immunomodulatory effects, inhibiting the capping of the viral genome, inhibiting the viral polymerase and/or inducing error catastrophes [93]. Ribavirin

has been approved for clinical use, including against hepatitis C virus infection and has been shown to inhibit JEV replication in vitro [94]. However, the oral administration of ribavirin for 7 days failed to improve the outcomes of JE patients in a

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randomized, double-blind, human clinical trial conducted in India [95]. A variety of virus-infected cells have been shown to produce reactive oxygen species (ROS), which are associated with viral pathogenicity, for example, by inducing cellular apoptosis [96]. Several studies have demonstrated that JEV infections induce ROS-mediated neuronal cell/tissue damage, implying that antioxidants that neutralize ROS are possible antiviral drugs against JEV infection [97]. To date, several antioxidants have been investigated for the treatment of JEV infection. Minocycline, a semisynthetic tetracycline antibiotic, displays neuroprotective properties against various diseases [98]. Treatment with minocycline was shown to reduce ROS production in JEV-infected cells, preventing cell death in vitro [99]. The intraperitoneal administration of minocycline reduced neuronal cell damage and provided complete protection against lethal JEV challenge in mice when the treatment was initiated 1 day after JEV infection [100], and even when the treatment was initiated after disease onset, the administration of minocycline provided partial protection in mice [100]. The intraperitoneal administration of another antioxidant arctigenin, a phenylpropanoid dibenzylbutyrolactone lignin, provided complete protection in mice when treatment was initiated 1 day after JEV infection [101]. Treatment with fenofibrate, an agonist of PPAR-a and known to induce antioxidant responses, also reduced JEV replication in vitro, and the subcutaneous administration of fenofibrate provided 80% protection in mice when the treatment was initiated 4 days before JEV infection [102]. However, fenofibrate did not provide protection from death when the treatment was initiated 2 days before JEV infection [102]. Curcumin, a phenolic compound extracted from the rhizome of Curcuma longa L., is also known to be an antioxidant and was shown to reduce ROS production in JEV-infected cells and to prevent cell death in vitro [103]. Pentoxifylline, a methylxanthine derivative, was initially licensed to treat intermittent claudication and was then found to be effective against a variety of diseases, including infectious diseases [104]. When pentoxifylline was tested as an anti-JE drug, it inhibited viral replication in vitro, and, when it was intraperitoneally administered to mice immediately after JEV infection, it conferred complete protection against JE [105]. Although pentoxifylline probably inhibits virion assembly and/or virion release in the case of JEV infection, the detailed mechanism remains unclear [105]. Nitazoxanide (2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide) was originally developed to treat intestinal parasitic infections, but it also displayed broad-spectrum antiviral activities [106]. Nitazoxanide inhibits JEV replication in vitro in the early-to-mid-JEV replication cycle and its intragastric administration to mice 1 day after JEV infection provided 90% protection from death [107]. Specific nucleic acid-based antiviral drugs The discovery of RNA interference and the microRNA machinery, which inhibit transcription and translation, 3.2

8

respectively, after the recognition of complementary sequences [108], has provided opportunities for the development of novel types of antiviral drugs. Because nucleic acid-based antiviral drugs react highly specifically, they are considered powerful candidates for the treatment of JE. To date, smallinterfering RNA-based anti-JE drugs targeting the C, M, E, NS1, NS3, NS4B and NS5 genes have been examined both in vitro and in vivo [109-113]. Although many of these candidates provided partial or complete protection in mice, they must be administered simultaneously with or before JEV infection [109-113]. The specificity of nucleic acid-based antiviral drugs is considered one of their benefits, but these drugs may not recognize multiple strains or genotypes because they are so specific. Candidate drugs containing a single microRNA polycistron with repeated NS1-targeting sequences or nine distinct targeting sequences for genome regions highly conserved among JEV genotypes inhibited the viral replication of genotypes I and III in vitro and were theoretically predicted to be effective against genotypes II and IV [114]. However, another obstacle to the use of nucleic acid-based antiviral drugs is that these drugs must be delivered into the cells in which the virus is replicating. After the onset of JEV infection, JEV replicates within the CNS, so most of the in vivo evaluations described above require the highly invasive intracranial delivery of the drugs [109,110,112,113]. To overcome this problem, joining the small-interfering RNA to a short peptide derived from a rabies virus glycoprotein that specifically interacts with the acetylcholine receptor expressed on neuronal cells was tested [115]. After the intravenous administration of this antiviral drug, it was successfully delivered into the brain tissue, suppressed the target gene expression and conferred partial protection against lethal JEV challenge [115]. Using similar approaches, the targeted delivery of drugs to dendritic cells and macrophages has been achieved [116,117]. Peptide nucleic acids (PNAs), synthetic nucleic acid derivatives with noncyclic peptide-like backbones carrying side chains that contain heterocyclic bases, are known to bind to their complementary sequences with high specificity, inhibiting their translation [118]. Several PNA-based antiviral drugs have been evaluated since their discovery. PNAs targeting the UTRs of positive-sense and negative-sense viral genomes were examined as potential anti-JE drugs [119]. To ensure their efficient uptake into cells, the PMAs were conjugated with a cell-penetrating peptide [119]. The PNAs targeting UTRs inhibited viral replication in vitro, probably by sterically interfering with genome cyclization [119]. Morpholino oligomers are single-stranded DNA analogs consisting of the same nitrogenous bases as DNA, in which each base is connected by a backbone composed of morpholine rings and phosphorodiamidate linkages. Morpholino oligomers also bind their complementary sequences causing steric interference [120]. Morpholino oligomers targeting the JEV UTRs have been evaluated [121,122]. For their efficient intracellular delivery, these morpholino oligomers were

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Potential chemotherapeutic targets for JE

conjugated with a cell-penetrating peptide or an octaguanidinium dendrimer [121,122]. The intraperitoneal or intracranial administration of these morpholino oligomers, initiated immediately after or before JEV infection, conferred partial protection against JE in mice [121,122]. Viral replication cycle-based antiviral drugs As shown in Figure 3, the JEV replication cycle consists of viral binding to its receptor, virus internalization, membrane fusion, RNA replication, viral protein synthesis, virion assembly and transport/release. Theoretically, each step is a potential target for antiviral drug development. Several distinct approaches have been used to inhibit the initial binding of the virus to its receptor. Because heparan sulfate is considered a viral receptor, cell-free heparan sulfates and their derivatives were examined. These drugs inhibited JEV infection in vitro [123,124] and provided partial protection in vivo when they were intraperitoneally administered before JEV infection [125]. Similarly, a peptide known to bind the E protein inhibited JEV infection in vitro, probably by disrupting the interaction between the virion and cellular receptors [126]. Surfactant-modified nanoscale silicate platelets inhibited viral attachment by interacting electrostatically with the virus [127]. Partial protection was observed in mice when they were intraperitoneally administered nanoscale silicate platelets immediately after lethal JEV challenge [127]. Indirubin, which is derived from Isatis indigotica, blocked viral attachment in vitro and provided partial protection against JE when the intracranial treatment was initiated immediately after lethal JEV challenge [128]. Bovine lactoferrin and griffithsin inhibited JEV infection by binding to cell-surface heparan sulfates [129,130]. The intraperitoneal administration of griffithsin before JEV infection provided complete protection in mice [130]. Recombinant partial E proteins/peptides derived from domain III, which is important in viral receptor binding, inhibited JEV infection in vitro by interacting competitively with the viral receptors, and preinoculation with these antiviral drugs provided partial protection in mice [131,132]. Several antiviral drugs targeting the RNA replication step have been reported. Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), which contains a nuclease domain, displays anti-JE activity in vitro, and the RNase, RNA-binding and oligomerization properties of MCPIP1 are required for its anti-JE activity [133]. Kaempferol, a polyhydric flavonol derived from various plants, inhibits JEV replication by binding to the viral genome in vitro [134]. FGIN-1-27, cilnidipine, niclosamide and bispidine--amino acid conjugates displayed anti-JE activities in vitro, probably by inhibiting RNA replication [135,136]. Pokeweed antiviral protein, a plant-derived N-glycosidase ribosome-inactivating protein isolated from Phytolacca americana, inhibited JEV replication in vitro by the depurination of the viral genome [137]. The intraperitoneal administration of pokeweed antiviral protein to mice before and after the JEV infection provided them with partial protection against JE [137].

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3.3

SCH16, an N-methyl isatin b--thiosemicarbazone derivative, inhibited JEV replication in vitro at the level of early viral protein translation [138]. The intraperitoneal administration of SCH16 after intracranial JEV challenge and the oral administration of SCH16 after intraperitoneal JEV challenge conferred partial protection in mice [138]. A combination of SCH16 with ribavirin or mycophenolic acid displayed a synergistic anti-JE activity in vitro [139], although anti-JE activities of the individual compounds were observed [94,140]. In silico modeling-based antiviral drugs Recent advances in structural virology have contributed to the resolution of the structures of viral proteins. Among the JEV proteins, the crystal structures of the NTPase/helicase catalytic domain of NS3 [141], E [142] and NS5 proteins [143] have been reported. Because the NS3 and NS5 proteins are multi-enzyme proteins that are essential for efficient viral replication, and because the E protein plays important roles in receptor binding and membrane fusion, as described above, antiviral drugs targeting these proteins are expected to be effective. Computer-based antiviral drug screening and/or design based on the resolved or predicted structures of the NS3 protein have been undertaken [144,145]. Ivermectin, which is used as an anthelmintic drug, displayed anti-JE activities in vitro [146]. 4-Hydroxypanduratin A, a secondary metabolite of Boesenbergia pandurata Schult, was selected in silico to interrupt the interaction between the NS2B and NS3 proteins by binding to NS2B, although no in vitro and in vivo evaluations have been performed [147]. 3.4

Intravenous immunoglobulin The intravenous immunoglobulin (IVIG) prepared in endemic areas may contain neutralizing antibodies, and IVIG is also known to have anti-inflammatory properties. Treatment with high-dose IVIG has been shown to be effective against various infectious diseases, including Flavivirus infection [148]. The treatment of a single JE patient with IVIG, begun on day 6 of hospitalization, was shown to be successful, although whether the IVIG contained anti-JE antibodies was not mentioned [149]. Therefore, IVIG treatment for JE is considered to be effective. In a recent preliminary, randomized, double-blind, human clinical trial [150], no significant difference in patient outcomes was observed after IVIG treatment, but only 22 patients were enrolled in the study [150]. 3.5

Potential targets for antiviral drug development Of the steps in the JEV replication process in infected cells, the initial/early steps have been the main targets for anti-JE drug development. However, as described above, every single replication step shown in Figure 3 is a theoretical target. Antiviral drugs are administered to patients who display symptoms. Therefore, anti-JE drugs must be effective in cells in which the virus is replicating by blocking intracellular viral replication, although antiviral drugs that inhibit viral 3.6

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expansion into naı¨ve cells by blocking the receptor-binding step are also considered important. Recent studies have shown that several host factors are involved in JEV replication, probably interacting with the viral genome directly or indirectly [51-58]. These interactions with host factors should be rational targets for anti-JE drugs. RNA replication occurs at virus-induced membrane structures [48,49]. In other flaviviruses, the NS proteins, particularly NS4A and NS4B, have been implicated in the formation of these characteristic membrane structures [151-153]. Therefore, drugs that inhibit the formation of these membrane structures might be considered potentially effective against JE, although there is no firm evidence that the virus-induced membrane structures are essential for viral replication. During the RNA replication step, protein--protein interactions between NS3 and NS5 are observed, in addition to the binding of these individual proteins to the viral genome [33]. The structures of these viral enzymes have also been resolved in West Nile virus (WNV) and dengue virus (DENV), and highthroughput strategies are available to screen drugs directed against these viruses using replicon-harboring cells. Therefore, many antiviral candidate drugs have been reported that inhibit these viral proteins by several mechanisms [154,155]. Identifying antiviral drugs that inhibit the enzymatic activities of these proteins is thought to be straightforward, and blocking these protein--protein interactions is another potential target of drug development. Only a few nucleic acid-based antiviral drugs targeting the UTR have been shown to inhibit genome cyclization, which is essential for efficient viral replication [119,121,122]. Therefore, identifying chemical compounds that interfere with genome cyclization are another goal of drug development. NITD-451, an anti-DENV drug, has been shown to inhibit viral translation both in vitro and in vivo [156]. Because this compound is thought to inhibit the translation of other flaviviruses [156], its potential action against JE should be evaluated. Similarly, lovastatin, which inhibits intracellular cholesterol synthesis, is reported to reduce DENV viremia in mice [157]. The importance of intracellular cholesterol for JEV replication has been demonstrated in vitro [158], so cholesterol and/or its synthesis are considered possible targets for anti-JE drugs. Glycosylation is essential for several viral proteins and glucosidase inhibitors have shown antiviral effects against flaviviruses, including JEV, in vitro [159]. Recently, celgosivir was evaluated for the treatment of patients with dengue infection in a randomized, double-blind, proof-ofconcept, human clinical trial [160]. Unfortunately, no significant improvement in the outcomes of the celgosivir-treated group was seen [160]. As an alternative strategy, a combination of a glucosidase inhibitor and ribavirin was evaluated and exerted a synergistic antiviral effect [161], suggesting that glycosylation can be a target of antiviral drugs. The detailed molecular mechanisms of virion assembly/ encapsidation remain largely unknown, but the C protein contains two positively charged domains at its N and C 10

termini, which probably interact with the viral RNA genome, and the Flavivirus C proteins are known to form homodimers [22]. Interestingly, ST-148, which displayed anti-DENV activity in mice [162], was recently found to interact with the DENV C protein [163] and is thought to inhibit capsid assembly and disassembly by stabilizing the self-interaction of C protein [163]. These studies demonstrated that a C-protein-targeting antiviral drug inhibited both viral entry (uncoating) and assembly. The development of drugs targeting the viral immune evasion mechanism has lagged far behind the development of antiviral drugs that target the intracellular viral replication mechanism. As described above, JEV uses several strategies to evade the host immune responses (Figure 3). To block IFN signaling, the NS4A and NS5 proteins inhibit the JAK/ STAT pathway by blocking the phosphorylation of the STAT proteins or TYK2 [63,64]. Although the region of NS4A responsible for this property has not been identified, the N-terminal region of NS5 is the region involved [63]. Therefore, these properties of the NS4A and NS5 proteins can be targeted. The NS2A protein is reported to inhibit PKR activation, and the 33th residue of NS2A is known to be essential [65]. Therefore, antiviral drugs targeting this residue are other potential candidate drugs for the treatment of JE. Autophagy is reported to be induced in JEV and to be involved in its efficient replication [70], whereas this is not true of WNV [164]. The autophagy-inducing peptide, Tat--beclin1, has been shown to reduce mortality in WNVinfected mice [165]. Although the function of autophagy in JEV differs from that in WNV, this study proposed that autophagy could be another antiviral drug target. Drugs that block the induction of autophagy may increase IFN production, resulting in stronger host antiviral responses. 4.

Conclusion

Because only supportive care is currently available for patients infected with JEV, the development of effective anti-JEV drugs is an urgent issue. Although numerous candidate drugs have displayed anti-JE activities in vivo, only three human clinical trials of such drugs have been completed, and sadly, none of the candidates showed an improved outcome. Because most infections are asymptomatic and preventive vaccines are available, the development of an antiviral drug for JEV infections might not be a commercially attractive target. However, JEV infection remains a major public health concern in many Asian countries, particularly in developing countries. Effective and inexpensive antiviral drugs are urgently required by these countries, together with increased vaccine coverage. Recent advances in our understanding of the detailed JEV replication mechanism, its immunology and structure should allow the identification of novel antiJE candidate drugs. The strategies used to develop antiviral drugs for other flaviviruses could also contribute to this work and should be incorporated into these projects.

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Therefore, many candidate drugs are expected to be evaluated in human clinical trials in the near future.

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5.

Expert opinion

JEV infection is a major public health threat in Asian countries. Epidemiological data have shown that the number of cases of JE decreased dramatically with the introduction of these vaccines into routine immunization programs in several countries, indicating that JE is a vaccine-preventable disease. However, many endemic countries, and particularly developing countries, suffer from low vaccine coverage. The decision of the GAVI Alliance to offer financial support to immunization campaigns and the inclusion of JEV vaccines in routine immunization programs is expected to increase vaccine coverage. Effective therapeutic antiviral drugs are required to reduce the disease burden in the current situation. Unfortunately, only supportive care is currently available to JE patients. Although several candidate drugs have been evaluated in clinical trials, no significant improvement in patient outcomes was seen. Many other candidate anti-JE drugs have been developed, and some of them inhibit viral replication in vivo. However, most are only effective when the treatment is initiated before JEV infection or immediately after infection. In the clinical situation, antiviral drugs are administered to patients developing symptoms, not before JEV infection or immediately after infection. Therefore, antiviral drugs that are effective when administered after the disease manifests are required. Combining multiple drugs is an interesting approach to the production of ideal anti-JE drugs. The combination of SCH16 (inhibits translation) with ribavirin (inhibits host inosine monophosphate dehydrogenase) or mycophenolic acid (inhibits host inosine monophosphate dehydrogenase) has shown synergistic effects [139]. The combination strategy has been employed in anti-DENV drug development using a glucosidase inhibitor with ribavirin [161]. This combination strategy has another benefit, in addition to its synergistic antiviral effect, because the dose of each antiviral drug can be reduced, while still achieving the same efficacy as the single drug, so its cytotoxicity (adverse effects) is reduced in the combination strategy. Because only combinations of nonspecific antiviral drugs have been examined so far, further combinations of multiple drugs from different drug categories must be evaluated to identify the best formulations. Numerous other compounds have also displayed anti-JE activity but have not yet been examined in vivo. For example, several flavonoids derived from natural sources have been shown to inhibit JEV replication and DENV infection [166,167]. These compounds must be evaluated in vivo, particularly in combination strategies. There is a major obstacle to treating JE patients. Because JEV invades the CNS, causing encephalitis, anti-JE drugs must penetrate the blood--brain barrier (BBB) and/or the

blood--cerebrospinal fluid barrier (BCSFB) to reach the CNS. The BBB and BCSFB are composed of tight junctions formed by capillary endothelial cells and choroid plexus cells, respectively [168]. Under physiological conditions, the BBB and BCSFB are essential for the homeostasis of the CNS and protect the brain from invasion by microbes. Therefore, the BBB and BCSFB act as the first line of the innate immune responses in the CNS. However, the BBB and BCSFB also prevent the delivery of antimicrobial drugs into the CNS. Increasing the doses of these drugs to ensure their delivery to the CNS may also increase the risk of adverse effects. Therefore, together with the development of effective antiJE drugs, the development of efficient drug delivery methods to the CNS is essential. In general, only small lipophilic chemicals can efficiently penetrate the BBB and BCSFB, although receptor-mediated and efflux transporter-mediated pathways are available for other molecules [168]. To overcome this obstacle, a number of approaches have been investigated, and among them, nanocarriers have been one of the most studied strategies [169]. For example, liposomes, nanoparticles consisting of polylactide homopolymers and PEGylated solid lipid nanoparticle have been used as carriers of drugs into the CNS [169]. These nanocarriers penetrate the BBB and BCSFB efficiently via endocytosis/transcytosis [169]. Furthermore, recent progress in the development of these nanomedicines has shown that drugs can be delivered to specific cell types when conjugated with specific molecules. For example, aptamers, which are single-stranded DNA or RNA molecules, recognize their target molecules like antibodies do [170], are relatively small and stable and are not immunogenic. Therefore, the conjugation of drugs with aptamers is considered a promising celltargeting strategy [170]. Although several nucleic acid-based anti-JE drugs with modifications that ensure intracellular delivery have been evaluated [115,119,121,122], others have not utilized these strategies. Therefore, efficient CNS delivery systems and/or specific-cell-targeting systems must be incorporated into the development of current and future anti-JE drugs. In the past decade, advances in basic JEV research, and in immunology, structural virology and molecular biology, have identified new antiviral molecular targets and new antiviral strategies. Therefore, in the coming decade, these research fields and potentially other new fields of research may provide new targets for anti-JE drug development and/or novel types of antiviral drugs. In addition to the development of anti-JE molecules themselves, efforts must be made to develop strategies for the efficient delivery of these drugs to the CNS and specific cell types. Efficient drug delivery to infected cells in vivo can reduce their toxicity, and thus allow the doses administered to be increased. If this is achieved, anti-JE drugs that have shown sufficient anti-JE activities in vitro but not in vivo should be reevaluated. Therefore, it is anticipated that specific effective treatments for JE will be established in the near future, and that JE will become a truly controllable

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disease worldwide, using both effective vaccines with high coverage rates and effective specific treatments.

Acknowledgments The authors thank Satomi Yoshida for her technical assistance in drawing the figures. They also thank the Edanz Group Ltd for English proof-reading.

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Tomohiro Ishikawa1 & Eiji Konishi†2,3 † Author for correspondence 1 Assistant Professor, Dokkyo Medical University, School of Medicine, Department of Microbiology, 880 Kitakobayashi, Mibu-machi, Shimotsuga-gun, Tochigi 321-0293, Japan 2 Mahidol University, BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, 420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand 3 Professor, Osaka University, Research Institute for Microbial Diseases, BIKEN Endowed Department of Dengue Vaccine Development, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan Tel: +66 2 354 5981; E-mail: [email protected]

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Affiliation

Potential chemotherapeutic targets for Japanese encephalitis: current status of antiviral drug development and future challenges.

Japanese encephalitis (JE) remains a public health threat in Asia. Although several vaccines have been licensed, ∼ 67,900 cases of the disease are est...
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