key: cord-0937309-hnuogpqn
authors: Acharya, Dhiraj; Bai, Fengwei
title: An Overview of Current Approaches Toward the Treatment and Prevention of West Nile Virus Infection
date: 2016-05-18
journal: West Nile Virus
DOI: 10.1007/978-1-4939-3670-0_19
sha: 96fa7b0201a495e59794b2b1c2e8cd794d1ba413
doc_id: 937309
cord_uid: hnuogpqn

The persistence of West Nile virus (WNV) infections throughout the USA since its inception in 1999 and its continuous spread throughout the globe calls for an urgent need of effective treatments and prevention measures. Although the licensing of several WNV vaccines for veterinary use provides a proof of concept, similar efforts on the development of an effective vaccine for humans remain still unsuccessful. Increased understanding of biology and pathogenesis of WNV together with recent technological advancements have raised hope that an effective WNV vaccine may be available in the near future. In addition, rapid progress in the structural and functional characterization of WNV and other flaviviral proteins have provided a solid base for the design and development of several classes of inhibitors as potential WNV therapeutics. Moreover, the therapeutic monoclonal antibodies demonstrate an excellent efficacy against WNV in animal models and represent a promising class of WNV therapeutics. However, there are some challenges as to the design and development of a safe and efficient WNV vaccine or therapeutic. In this chapter, we discuss the current approaches, progress, and challenges toward the development of WNV vaccines, therapeutic antibodies, and antiviral drugs.

may cause injury and death of neurons with various clinical manifestations, such as encephalitis , meningitis , fl accid paralysis, persistent neurologic sequelae , and possibly death, particularly in the elderly and immunocompromised individuals [ 1 , 10 , 11 ]. WNV strongly activates host immune responses, which play important roles in controlling viremia, viral dissemination to the central nervous system (CNS), and recovery from the disease [ 11 ] . However, the mechanism of WNV pathogenesis , including its tropism to neurons, CNS invasion, and viral or host factors that contribute to imbalance between viral pathology and host immunity still remain poorly understood.

Although WNV was fi rst discovered in Uganda in 1937, it had been considered as a minor public health concern until its fi rst appearance in the USA in 1999 [ 12 ] . Since then, it has dramatically spread to all the continental states of the USA and became an endemic disease throughout North America within a few years [ 13 -16 ] . In the USA alone, there have been over 40,000 reported cases of WNV between 1999 and 2014, of which ~45 % were classifi ed as neuroinvasive and claimed lives of nearly two thousand people [ 17 ] . However, the actual WNV burden is likely much higher than previously thought because only about 20 % of infected individual develop a clinical WNV disease [ 13 ] . It has been estimated that over three million individuals have been infected with WNV in the USA, of which about 780,000 had a symptomatic disease [ 18 ] . WNV also has potential to develop unusual clinical manifestations [ 19 -21 ] and may involve in renal diseases [ 22 , 23 ] , myasthenia gravis [ 24 ] , and myocarditis [ 25 ] , suggesting that the range and severity of WNV disease may be even worse than previously believed. Importantly, increasing numbers of WNV outbreaks during the last 15 years have been associated with greater number of neuroinvasive cases and a higher rate of fatalities [ 16 , 17 ] . However, no vaccine or antiviral therapeutic is currently available, which limits current treatments to only supportive care measures, such as intravenous fl uids, antipyretics, respiratory support, and prevention of secondary infections. Considering the worldwide distribution of this virus and evidence of its potential to change in pathogenicity and transmission [ 26 -30 ] , there is an urgent need to develop safe and effective antiviral drugs or vaccines against WNV infection [ 31 ] . Intensive research during past decades has made signifi cant progress in the design and development of several treatment and prevention methods for WNV infection (reviewed by [ 32 -34 ] ). Here, we discuss the current approaches and recent progress toward the development of vaccines, therapeutic antibodies, and antiviral drugs against WNV infection in humans.

WNV is a spherical virus with 50 nm in diameter, which comprises an icosahedral nucleocapsid surrounded by a lipid envelope [ 35 ] . The virus contains a single-stranded, capped, and plus-sensed RNA genome of approximately 11 kb in size. The viral genome encodes a polyprotein precursor, which undergoes posttranslational processing by cellular and viral proteases to generate three structural proteins (capsid [C] , premembrane [PrM] , and envelope [E]), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins form virion structure, whereas the non-structural proteins play essential roles in the replication of viral genome, assembly of virion, and viral pathogenesis [ 36 -38 ] . Thus, the structural and non-structural proteins of WNV may be potential targets for developing vaccines and antiviral therapeutics (Table 1 ) .

In a WNV virion structure, C protein encapsulates viral genomic RNA to form a nucleocapsid that is enveloped by a lipid bilayer into which trimmers of prM-E heterodimers form the spikelike projections. Among these structural proteins, E protein mediates crucial roles in binding to cellular receptors, membrane fusion, and entry of WNV into host cells, making it a key target for the development of vaccines, neutralizing antibodies, and entry inhibitors . Crystal structure analysis has confi rmed that E protein folds into three structurally distinct ectodomains (EDs) termed EDI, EDII and EDIII [ 39 -43 ] . Among these, the EDIII consists of the major neutralizing epitopes and is an antigen of choice to elicit production of neutralizing antibodies [ 43 -46 ] . Based on the structural characterization of antifl aviviral monoclonal antibodies from both human and nonhuman primate, it appears that the epitopes of fl aviviral E protein are more complex and diverse than previously thought [ 47 -50 ] . In addition, mapping of B-cell and T-cell epitopes has led to the identifi cation of many immunodominant epitopes in both structural and nonstructural proteins of WNV [ 51 , 52 ] .

Among the non-structural proteins , NS3 and NS5 are bestcharacterized, multifunctional proteins, both of which contain enzymatic activities that are essential for viral replication [ 53 -55 ] . Such enzymatic functions of NS3 and NS5 have received considerable attention as potential targets for antiviral drug development [ 34 , 54 ] . The NS3 protein contains two distinct functional domains. The N-terminal domain of NS3 (184 amino acid residues) has serine protease activity that requires a polypeptide cofactor NS2B for activation [ 54 , 56 , 57 ] . Recent X-ray crystallographic studies have shown that the conformation of β-loop of NS2B controls the substrate binding by NS2B/NS3 protease [ 58 , 59 ] . In contrast, the C-terminal domain of NS3 functions as an RNA helicase, [ 67 ] . Besides its function in viral replication , NS5 also plays a role in viral pathogenesis by antagonizing host's interferon response [ 28 ] . Other nonstructural proteins NS2A, NS2B, NS4A, and NS4B form the scaffold for the viral replication complex and also have roles in the replication of viral genome and host immune evasion [ 68 -70 ] . Mutations in NS4B protein may attenuate WNV and other fl aviviruses [ 68 , 69 ] . In addition, a recent successful clinical trial of a hepatitis C virus NS5A inhibitor suggests that targeting non-structural proteins may be an ideal strategy to develop therapeutics against other fl aviviruses, including WNV [ 71 ] . 74 -78 ] . In particular, the C-terminal EDIII of E protein that contains critical neutralizing epitopes is the major target for neutralizing antibodies against WNV infection [ 40 , 44 , 78 , 79 ] . In addition to humoral immune response, cell mediated immunity by CD4 + and CD8 + T cells play critical roles in recovery from WNV infections [ 80 -83 ] . Thus, the effi cient generation of vaccineinduced immunity against WNV may also require activating and shaping of multiple effectors of adaptive immune response by early innate signaling pathways [ 84 -86 ] .

Development of an effective vaccine requires multiple steps from design and development to rigorous evaluation of both safety and effi cacy (Fig. 1 ) . Since the biology of vaccine-induced immunity and principles in vaccine development and testing have been extensively discussed before, we will not discuss these topics here. Immunization of the laboratory animals and subsequent challenge of those animals with a pathogen under controlled conditions is the common method for early evaluation of vaccine effectiveness. Introduction of three mutations responsible for attenuation of JEV (SA14-14-2) in equivalent positions of WNV E gene further attenuated ChimeriVax-WN02. Similarly, DENV4 vaccine candidate (rDEN4Δ30), attenuated through a 30-nucleotide deletion in the 3′ untranslated region (UTR) of the viral genome , was further engineered to express WNV-NY99 prM and E [ 122 , 123 ] . After preclinical evaluation in mice, geese, and monkeys, rDEN4Δ30 showed strong immunogenicity in the clinical trial [ 94 ] . In addition, a chimeric DENV2 vaccine candidate expressing the WNV NY99 prM and E proteins has been shown to protect mice from infection with WNV NY99 strain [ 124 ] . Another recombinant WNV vaccine based on infl uenza vaccine (FLU-NA-DIII) was developed by cloning DIII of WNV E into the N-terminal region of neuraminidase of infl uenza virus. This vaccine candidate induced WNV-specifi c neutralizing IgG and protected mice against lethal WNV infection [ 125 ] . Similarly, a recombinant adenoviral vaccine vector (CAdVax-WNVII) expressing all three structural proteins (C, prM, and E) along with NS1 of WNV induced neutralizing antibodies in mice [ 

The most common approach to develop non-replicating inactivated viral vaccines is to inactivate entire virus particles by using chemicals. A formalin-inactivated WNV vaccine based on WNV-NY99 strain was the fi rst successful veterinary vaccine (marketed by Pfi zer as West Nile Innovator ® ) licensed in 2003 [ 127 ] . Another veterinary WNV vaccine using killed virus was also licensed by USDA (marketed by Boehringer Ingelheim as Vetera™ WNV). Recently, an inactivated WNV vaccine (WN-VAX) based on WNV NY99 protects mice against lethal WNV infection and exhibits immunogenicity in monkeys [ 128 ] . In addition to WNV NY99, formalin inactivation of WNV IRS98 strain induces neutralizing antibody and protects immunized geese [ 129 ] . As an alternative to traditional formalin-based vaccines, a novel hydrogen peroxide (H 2 O 2 ) inactivation approach has been recently used to produce a wholevirus vaccine against WNV [ 130 , 131 ] . Mice immunized with H 2 O 2 -inactivated WNV vaccine candidate developed high serum neutralizing titers , and offered complete protection of vaccinated mice against lethal WNV challenge [ 130 ] . One of such H 2 O 2 inactivated vaccine (HydroVax-001) has been recently introduced into phase I clinical trial. Although inactivation of virulent WNV virus strain has been successfully achieved by chemical-inactivation method [ 127 , 129 ] , use of a naturally attenuated Kunjin strain of WNV [ 132 , 133 ] , or chemically synthesized virus by cDNA system [ 134 ] as starting material has also been proposed.

Several studies demonstrated that soluble recombinant protein or VLP based approach could serve to develop WNV vaccines [ 135 -139 ] . VLP are specialized subviral particles that lack of viral genome and solely contain viral structural proteins [ 140 , 141 ] or express viral proteins on envelope membranes [ 142 , 143 ] . Different vectors and production system were evaluated for development of various subunit vaccines against WNV. For example, a recombinant truncated form of WNV E protein produced in Escherichia coli induced neutralizing antibodies and protected mice from lethal WNV challenge [ 46 , 144 ] . In addition, a recombinant truncated WNV E protein produced in the SF+ insect cells via baculovirus infection induced neutralizing antibodies and protected mice and hamsters from WNV infection [ 145 ] . Recombinant baculovirus was also used to express WNV prM and E proteins in mammalian cells under the CMV promoter, with or without vesicular stomatitis virus glycoprotein (Bac-G-prM/E). Such vaccines induced robust immune responses when inoculated in mice and produced both neutralizing antibodies and infl ammatory cytokines [ 146 ] . In a recently proposed novel approach, known as pseudotyping, a retrovirus Gag polyprotein forms a VLP scaffold to display the ectodomain of human membrane glycoprotein (CD16) that was fused to the high affi nity IgE receptor gamma chain (RIgE).

Using this retrovirus based VLPs platform, a WNV vaccine was generated by replacement of the CD16 ectodomain in CD16-RIgE glycoprotein with EDIII of WNV, which induced neutralizing antibodies in mice [ 139 ] .

A recombinant E protein of WNV-NY99 produced in Drosophila S2 cells (WNV-80E, developed by Hawaii Biotech) is the only WNV subunit vaccine candidate that has been tested in phase I clinical trial. Although preclinical studies revealed WNVspecifi c neutralizing antibody responses in vaccinated animals [ 136 , 147 , 148 ] , the immunogenicity of this vaccine in humans was low. To increase immunogenicity, conjugation of recombinant proteins with nanoparticles or pathogen associated molecular patterns (PAMPs) as carrier/adjuvant have been tested. For instance, a recombinant WNV E protein administered with unmethylated CpG oligonucleotide adjuvant or loaded onto CpG-modifi ed nanoparticles strongly activated dendritic cells and lymphocytes and elicited Th1-dominant immune responses by producing high titers of IgG2a and IgG2b in immunized mice [ 149 , 150 ] . Similarly, mice injected with DIII of WNV E conjugated with bacterial fl agellin (STF2∆.EIII) [ 137 ] or VLP derived from bacteriophage AP205 engineered to express DIII of WNV E (DIII-C-AP205) [ 138 ] also signifi cantly increased neutralizing antibody production and protected the immunized mice.

Despite the intensive efforts in development of WNV vaccines, only a few reached the clinical trial stages. Of those in clinical trials, most candidate vaccines fail to demonstrate effi cient immunity and safety. Development of new tools for antigen screening, expanded understanding of immunological correlates of vaccine inducedimmunity, and discovery of novel adjuvants for vaccine delivery may facilitate the design and the development of WNV vaccines . For example, knowledge of genomic information and bioinformatics has been used for in silico identifi cation of candidate antigens and development of vaccines by a novel method called "reverse vaccinology" [ 151 , 152 ] . This comprehensive tool can quickly identify all potential antigens coded in the genome and may be used to develop a novel viral vaccine [ 153 ] . Development of vaccine against group B streptococci proved the potential of this approach [ 151 ]. Similarly, a "structural approach" that improves antigenicity of vaccines by rational designing has been developed by utilizing the knowledge of immunology, structural biology, and bioinformatics [ 154 ] . In addition, increased understanding of immunogenetics and role of environmental and host factors that determine the variation of vaccine immunity may offer new approaches to design a more effective vaccine against WNV infection in humans.

Therapeutic monoclonal antibodies (mAbs) or hyperimmune sera have been successfully used for prophylaxis of a number of infectious and noninfectious diseases, including WNV infection. In recent years, the number of mAbs in preclinical development and clinical trials has been increased signifi cantly [ 155 ] . So far, nearly 50 mAbs have been approved for therapeutics by US FDA, including a humanized mAb Synagis (palivizumab) for preventive use against respiratory syncytial virus ( WNV infection induces a potent humoral immune response, which is essential in controlling viremia and limiting WNV dissemination to the CNS [ 72 , 73 ] . Hyperimmune sera, γ -globulin, or affi nity-purifi ed antibodies harvested from WNV-infected humans and animals protect both wild-type and immunocompromised mice from WNV challenge in laboratory conditions [ 164 -166 ] . In addition, WNV patients who received antibodies from the WNV seropositive donors recovered from WNV infection [ 167 -171 ]. These studies not only encouraged the efforts toward the development of human or humanized monoclonal antibodies against WNV, but also led to the discovery of several potent monoclonal antibodies that showed effi cient protection of mice and hamsters from WNV infection [ 172 -175 ] . Among these, a humanized anti-WNV mAb (Hu-E16) that binds to a highly conserved epitope in WNV E protein blocks viral fusion and provide post-exposure therapeutic potential [ 172 ] . This antibody is currently being assessed for its potential use as a WNV therapeutic antibody [ 176 ] . The phase I clinical trial showed that another humanized recombinant antibody targeting E protein of WNV (known as MGAWN1) has a good safety and tolerance profi le in healthy humans [ 177 ], however, the phase II trial to assess its effi cacy in WNV infection failed due to poor enrollment of participants. Besides the development of whole antibody, recombinant fusion proteins are also generated from single-chain antibody fragment of the variable region. Such antibody fragments that target E protein may be potential candidates for immunoprophylaxis and therapy of WNV infections. A recombinant human single-chain variable region antibody fragments (Fv-Fc) fusion protein has a protective role against WNV infection in mice [ 178 ] .

One of the potential limitations of this approach is antibodydependent enhancement (ADE), a phenomenon by which infection of some viruses is enhanced by virus-reactive antibodies resulting in more effi cient virus entry through Fc receptor-mediated pathways. This phenomenon plays a role in the pathology of severe dengue infection and has also been observed in WNV in vitro [ 179 ] . Although the role of ADE in WNV disease in unclear, the development of a therapeutic antibody against WNV should address this potential issue. Another limitation of antibody-based therapeutics is high production cost, which limits mAb scalability. Producing therapeutic proteins, including antibodies in plants, may be a promising solution. Feasibility of this approach has been affi rmed by the successful production of anti-WNV monoclonal antibody Hu-E16 in plants ( Nicotiana benthamiana ) (MAb-pE16) [ 180 , 181 ] . The plant-derived MAb-pE16 confers a potent neutralizing activity in vitro without ADE, effi ciently binds to complement and Fc receptors, and protects mice against lethal WNV-challenge with similar potency as their mammalian-cell counterparts [ 180 , 182 , 183 ] .

Antibodies employ several mechanisms to control WNV and other viral infections, including blockage of viral entry, Fc-dependent viral clearance, complement-mediated viral lysis, and antibodydependent cytotoxicity of infected cells. Most of the current researches in the development of therapeutic antibodies against WNV are designed and tested for effi cient neutralization potential [ 184 , 185 ] . Increased understandings of the biology of antibody Fc regions, in particular, the roles of glycan in Fc mediated functions may facilitate the design and development of high-quality antibody through glycoengineering [ 186 , 187 ] . Such engineering of antibody Fc region may be used to overcome ADE, modulate pharmacokinetics, and enhance Fc mediated effector functions, such as enhancement of antibody-dependent cell mediated cytotoxicity (ADCC), complement binding, and phagocytosis [ 188 ] .

Recent technological advancement not only in development, production, and purifi cation but also in ease of achieving desirable quality, effi cacy, and safety required for the FDA approval makes monoclonal antibodies a promising therapeutic option. Thus, monoclonal antibodies may prove useful for WNV prophylaxis and therapy particularly in the elderly and immunocompromised individuals with limited ability to respond to a vaccine . To meet its therapeutic goal, a controlled clinical trial of therapeutic antibody should ensure its prophylactic and therapeutic effi cacy along with optimal dose and timing of administration across the range of patient groups.

Development of effective therapeutics have been successful in treating many viral diseases including infl uenza, HIV-1, hepatitis C virus (HCV), and hepatitis B virus (HBV). WNV causes transient viremia in human and animal models that is associated with Treatment and Prevention Approaches for WNV its dissemination to brain and development of more severe disease [ 73 , 189 -192 ] , suggesting that reducing viral loads by an antiviral drug during the early phase of infection may offer effi cient control of WNV or lessen the chances of progression to neuroinvasive diseases. In addition, antiviral drugs are particularly useful for the elderly and the immunocompromised patients who may fail to develop effi cient vaccine -induced immunity. Recent progress in the structural characterization of WNV and other fl aviviruses broadens the understanding of WNV biology and provides a foundation for the development of small molecule inhibitors for WNV therapeutics [ 34 , 193 ] . In addition, better understanding of the pathogenesis of WNV and other fl aviviruses has offered new opportunities for designing many different classes of promising antiviral therapeutics by targeting both viral replication and the host cell metabolism.

The development of an antiviral drug is a multistep process that takes years before it reaches the market. A general overview of a drug development process is outlined in Fig. 2 . Hit-to-lead is an initial stage in a drug discovery, where small molecule hits are screened and further evaluated to identify promising lead compounds with a therapeutic potential. Recent progress in the development of multiple approaches for designing, screening, identifi cation, and validation of hit compound (reviewed by [ 194 -199 ] ) have witnessed growing interests in the fi eld of drug development. Signifi cant progress in structural and functional characterization of both structural and nonstructural proteins of WNV and other fl aviviruses has facilitated identifi cation of therapeutic targets and hit-to-lead screening. For example, characterization of pseudo-atomic structure of mature and immature WNV [ 35 ], atomic resolution structure of WNV and other fl aviviral protein by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy [ 39 , 41 , 48 , 200 -203 ] , and structural characterization of binding of a neutralizing monoclonal antibody to E protein of WNV [ 48 ] have greatly increased our understanding of both structural and functional aspects of potential therapeutic targets. Two approaches have been commonly used for small molecule inhibitor screening include target-based approach and cell-based approach. For target-based screening, several methods can be applied, including enzyme activity-based screening, fragmentbased screening, affi nity-based screening, structure-based rational [ 197 , 198 , 204 -207 ] . In contrast, cell-based approaches use viral infection and replication -dependent assays to identify inhibitors [ 208 ] . Each of these approaches presents their unique sets of merits and challenges . For instance, it is generally diffi cult to identify a target and also achieve specifi city by cell-based assay because such identifi ed inhibitors may potentially affect multiple steps of viral infection cycles and may target both viral and host proteins. Although inhibitors identifi ed by cell-based assay may prove useful as antiviral drug candidates, these compounds could also act nonspecifi cally; thus further elucidation of their mechanism of action is required. The target-based approaches are highly effi cient in screening process, however, an inhibitor screened by such method may require further modifi cation for effective cellular permeability and validation for its antiviral activity, selectivity and toxicity by using cell-based assays. In the fi nal stages, lead compounds are selected for in vitro and in vivo pharmacokinetic profi ling, including effi cacy, plasma stability (halflife), exposure, bioavailability, and preclinical toxicity before planning a clinical trial.

The most common strategies for antiviral drug development include blocking virus attachment or entry into host cells and inhibiting viral replication , either by targeting on viral components or host cells metabolism. There has been a signifi cant progress in development and testing of potential antivirals against fl aviviruses including WNV (reviewed by [ 34 , 209 , 210 ] ). Several natural and synthetic compounds, antiviral peptides and siRNAs have been identifi ed to target both structural and nonstructural proteins of WNV and evaluated for their potential therapeutic roles. Other approaches include targeting host cell metabolism and physiology and modulating host immune system by using antiviral cytokines as potential therapeutics against WNV. Current status and strategy used to develop antiviral drugs targeting WNV and other fl aviviruses are described below.

Several natural and synthetic compounds have been identifi ed to target both structural and non-structural proteins of fl aviviruses. Most of these compounds are designed to target DENV and some of them also show antiviral activities against WNV and other fl aviviruses. As these compounds are diverse in their chemistry, they are discussed below based on their modes of action.

The E protein of WNV and other fl aviviruses play key roles in viral entry into host cells by mediating viral attachment to host cell receptors and subsequent membrane fusion [ 35 , 41 , 211 , 212 ] . After binding to host cell receptors, WNV enters into cells through a clathrin-independent endocytosis process followed by a low-pHdependent viral uncoating in the endosome to release viral genome

into the cytoplasm for replication [ 213 ] . Inhibitors that disrupt the interaction of E protein with cell receptors or inhibit membrane fusion would be a potential antiviral against WNV. Two successful HIV drug Maraviroc (CCR5 antagonist) and Enfuvirtide (a peptide inhibitor) that respectively block viral entry [ 214 ] and membrane fusion [ 215 ] attest to this antiviral strategy. The fl aviviral E glycoprotein contains several functional sites such as a hydrophobic pocket, the receptor-binding domain and stem domain that may be targeted by inhibitors. Among these, the hydrophobic ligand-binding pocket in a hinge region between domain I and II of E protein plays an important role in low-pH-mediated membrane fusion process and is a unique target for developing smallmolecule inhibitors against fl aviviruses [ 216 , 217 ] . Various screening approaches were used to identify inhibitors against DENV and other fl aviviruses that bind into this hydrophobic pocket and interfere with the conformational changes of E protein [ 207 , 218 -221 ] , Most of these inhibitors were designed and tested against DENV, some of which were reported to exhibit antiviral activities against WNV [ 207 , 220 ] . However, these compounds failed in the further drug development due to their undesirable properties, such as low solubility and cytotoxicity.

The domain III of E glycoprotein that mediates receptor-binding can be potentially targeted by developing inhibitors that can disrupt the viral attachment to host cell receptors. Neutralizing antibodies against E protein have proven the potential of this strategy. Several compounds have also been shown to interfere with the binding of fl aviviruses to host cell receptors [ 222 -226 ] . However, lack of understanding of cell receptor for WNV has hampered the success of this approach. Identifi cation of cellular receptors for WNV and understanding of virus-receptor interaction may provide new opportunities to identify small molecule inhibitors that interrupt the binding of WNV to host cell receptors. Viral proteases are essential for WNV life cycle for they cleave the viral polyprotein precursors into functional proteins. Successful development and licensing of protease inhibitor against HIV-1 [ 227 , 228 ] and HCV [ 229 , 230 ] provides the proof of concept and feasibility for similar targeting of proteases of other viruses. The N-terminal domain of fl aviviral NS3 (amino acids 1-169) has serine protease activity whereas a hydrophobic region of NS2B protein serves as cofactor to activate the enzymatic activity of NS3 [ 53 , 231 , 232 ] . The NS3 protease of WNV processes the viral polyprotein precursor into structural and non-structural proteins and disruption of this activity is lethal for WNV [ 54 , 57 ] . In addition, WNV NS2B/NS3 protease can also cleave host proteins and may contribute to neuropathogenesis [ 63 ] . Recent progress in the expression of stable NS2B/NS3 and identifi cation of the highaffi nity substrate for this viral enzyme has promoted large-scale

Dhiraj Acharya and Fengwei Bai screening of protease inhibitors for WNV and other fl aviviruses . A wide range of assays, such as conventional enzyme-substrate based detection , HPLC, ELISA, and high-throughput fl uorescencebased detection methods have been developed for screening of viral protease inhibitors [ 233 -236 ] . Thus, NS3 protease is an attractive target for the development of antiviral against WNV and other fl aviviruses (reviewed by [ 237 ] ).

Except for aprotinin, a pancreatic trypsin inhibitor, most of the classical inhibitors of serine protease do not inhibit fl aviviral NS2B/ NS3 protease activity [ 238 , 239 ] . Although aprotinin is a potent inhibitor of fl aviviral protease, this compound was withdrawn from the market in 2008 due to safety issues [ 240 ] . To screen and identify small molecule inhibitors of fl avivirus protease, both high throughput screening and structure-based drug designing have been used. These strategies are based on the identifi cation of allosteric inhibitors that target the interface of NS2B-NS3 protease, or the active site of NS3 protease. The former strategy may overcome nonspecifi city of the latter due to the largely conserved active sites of the human and viral serine proteinases. A number of inhibitors for WNV NS2B/NS3 protease have been identifi ed by in silico docking or high-throughput screen using in vitro enzyme activitybased assays [ 34 , 59 , 206 , 241 -249 ] . However, most of these compounds failed to demonstrate potent antiviral activity in cell culture . Although a few compounds identifi ed by these approaches show anti-WNV activity in cell-based assays [ 62 , 206 , 242 ] , none of the inhibitors has progressed beyond the hit optimization stage. Discovery of NS2B/NS3 protease inhibitors has been hampered largely due to the diffi culties in obtaining co-crystal structures of inhibitor-protease complexes. Moreover, because of the weak binding affi nity of NS2B/NS3 active site due to its fl at and charged nature, the design of potent small molecule inhibitors by structurebased method becomes diffi cult [ 53 , 242 , 250 , 251 ] .

The helicases are enzymes that unwind nucleic acid by using energy derived from hydrolysis of NTP. The C-terminal domain of NS3 of WNV contains helicase/nuclease activities and plays important roles in virulence and pathogenesis [ 252 -254 ] . High throughput assays that measure helicase activity by monitoring helicase-catalyzed strand separation in real-time by using radioactive or fl uorescent-labeled oligonucleotides have been developed to screen helicase inhibitors [ 255 -258 ] .

Several small molecule inhibitors targeting helicase of HCV and HIV-1 have been developed [ 259 ] . By using the substratebased assay, a few compounds have been identifi ed and evaluated in vitro against NTPases/helicases of WNV and other fl aviviruses [ 260 -262 ] . However, inhibitory effects of WNV helicase by these compounds are specifi c to either DNA or RNA substrate. For example, a compound named 4,5,6,7-tetrabromobenzotriazole NS3 Helicase/Nuclease Inhibitors Treatment and Prevention Approaches for WNV 268 (TBBT), a halogenated benzotriazole, inhibits NS3 helicase, but not NTPase activity [ 260 ] . A series of ring-expanded nucleoside/ nucleotide analogs (RENs) also inhibit NTPases/helicases activities of fl aviviruses, including WNV, HCV, and JEV [ 263 , 264 ] , however, these compounds did not show any promising anti-WNV activity in cell culture . A nucleoside analog imidazo[4,5-d]pyridazine nucleosides [ 265 ] , and a broad-spectrum antiparasitic drug named ivermectin [ 266 ] inhibit NS3 helicase and also show anti-WNV activity in cell culture.

The RNA-dependent RNA polymerase (RdRp) activity of C-terminal NS5 protein of WNV and other fl aviviruses is an attractive target for developing antiviral agents [ 55 , 267 -269 ] . Two approaches used to target WNV RdRp include nucleoside inhibitors (NIs) or non-nucleosides inhibitors (NNIs). NIs (also known as type 1 inhibitors) are nucleoside/nucleotide analogs that target the active sites of the polymerase and generally compete with natural NTP substrates of RdRp to block their incorporation into viral genome during replication and lead to incomplete replication or mutations of viral genome. The success of NIs against several viruses including HIV-1, herpesviruses, HBV, and HCV has already proved the therapeutic potential of this class of compounds [ 270 -272 ] . In addition, NI generally displays broad-spectrum antiviral activities across related RNA viruses suggesting its potential as pan-fl aviviral therapeutics . Various cell-based and cell-free assays have been developed for high-throughput screening of fl aviviral RdRp inhibitors [ 273 -276 ] .

So far, several NIs that inhibit WNV, DENV, and other RNA viruses have been identifi ed [ 34 ] . For example, favipiravir (T-705; 6-fl uoro-3-hydroxy-2-pyrazinecarboxamide) and related compounds selectively inhibit viral RNA-dependent RNA polymerase and have potent anti-infl uenza activity [ 277 , 278 ] . This antiviral drug is currently being evaluated in clinical trials against infl uenza virus. In addition, favipiravir also blocks replication of many other RNA viruses, including WNV and are promising drug candidate against a broad range of RNA viral diseases [ 279 ] . Two other nucleoside analogs called 7-deaza-2′-C-methyl-adenosine and 5-aza-7-deazaguanosine (ZX-2401), which are the derivatives of triphosphates of 2′-C-methyl-adenosine and 2′-C-methyl-guanosine, respectively, are also broad-spectrum antiviral compounds targeting viral RdRp that inhibit DENV, HCV and WNV [ 280 -282 ] . Similarly, two other NI inhibitors , NITD-008 (beta-D-2′-ethynyl-7-deazaadenosine triphosphate) and NITD203 (3′,5′-O-diisobutyryl-2′-Cacetylene-7-deaza-7-carbamoyladenosine) inhibit all four of DENV serotypes and WNV.

In contrast to NI inhibitors , antiviral NNI inhibitors (also known as type 2 inhibitors) interfere with the function of viral polymerase by occupying its allosteric sites, thus preventing viral

Dhiraj Acharya and Fengwei Bai RNA synthesis. Analysis of RdRp crystal structure of WNV and DENV3 revealed a cavity that plays a critical role in viral replication , suggesting a potential target for screening of structurebased allosteric inhibitors [ 55 , 283 ] . N-sulfonylanthranilic acids derivatives identifi ed by high-throughput screening are examples of allosteric inhibitors of RdRp activity of DENV [ 284 ] . However, these compound were specifi c to DENV and did not show any activity against WNV RdRp. A recent study demonstrated that a conformational change occurred in DENV-3 polymerase after binding with an inhibitor [ 285 ] . However, a similar antiviral activity of NNI inhibitors targeting polymerase of WNV has not been reported yet.

Messenger RNA (mRNA) of WNV possesses a 5′ cap that plays important roles in stability of mRNA and its translation. The methyltransferase (MTase) activity of the N-terminal domain of NS5 is responsible for N-7 and 2′ O-methylation of the viral RNA cap [ 64 , 286 ] [ 287 ] . In addition, MTase activity is also responsible for evading host's antiviral interferon response and plays an important role in WNV pathogenesis [ 288 ] . Several structural and functional studies along with identifi cation of several potential inhibitors suggest that targeting MTase represents a novel approach for the development of novel therapeutics against WNV and other fl aviviruses [ 289 -294 ] . Flaviviruses MTase catalyzes sequential methylations of the viral RNA cap using S-adenosyl-L -methionine (SAM) as the methyl donor and contains a single binding site for SAM in its crystal structure [ 289 , 294 ] . In addition to MTase activity, binding of GTP has been shown in MTase domain of several members of fl avivirus [ 295 ] . Several assays have been developed for high-throughput screening of methyltransferase inhibitors by structural-based and ligand-based methods [ 296 , 297 ] . Rational design of SAM analogs has identifi ed several inhibitors targeting MTase activity of DENV and WNV [ 34 , 298 ] .

Nonspecifi c inhibition of host MTase is one of the potential drawbacks of SAM analogs. A specifi c inhibition of fl aviviral, but not host, MTase can be achieved by targeting a pocket near the SAM-binding site [ 290 , 298 ] . Two nucleoside analogs were identifi ed that potently inhibited the MTase of WNV without inhibiting human MTase. One of these compounds (GRL-003) showed antiviral activity against WNV in cell culture [ 299 ] . In addition, several screening studies against YFV and DENV NS5 have identifi ed hits targeting MTase activity, some of which showed antiviral activity against WNV in cell culture [ 204 , 300 , 301 ] . However, an extensive multistage molecular docking approach to screen a library of about 5 millions of commercial compounds against two active sites of DENV MTase/GTase failed to identify any specifi c hits [ 302 ] . Recently, 5′-silylated nucleoside scaffold derived from 3′-azidothymidine (AZT) demonstrated antiviral activity against WNV and DENV, which binds MTase [ 303 ] .

Several potential tools, including rational design and phase display library, have been developed for high-throughput screening of specifi c antiviral peptides [ 304 , 305 ] . Enfuvirtide, a 36-aminoacid peptide based on the stem region of the HIV gp41, exemplifi es an effi cient antiviral peptide currently in clinical use [ 306 ] . Thus, antiviral peptides may serve as a novel therapeutic measure against WNV. Several antiviral peptides targeting both structural and non-structural protein of WNV and other fl avivirus have been identifi ed.

Targeting WNV E protein by antiviral peptides is a potential strategy that blocks virus attachment and entry into the host cells. Several short antiviral peptides (13-16 amino acid residuals) that bind to WNV E protein have been identifi ed by screening of a murine brain cDNA phage display library [ 307 ] . One of those peptides (P9) reduces viremia and fatality after WNV infection in mice. P9 can effi ciently penetrate the murine blood-brain barrier , implying that it may have antiviral activity in the CNS [ 307 ] . Similarly, a peptide inhibitor (WN83) targeting domain II of WNV E protein designed by using a physicochemical algorithm approach potently inhibits WNV infectivity [ 308 ] . Another peptide designed to target domain II of DENV E shows antiviral activity against both DENV and WNV [ 308 ] . In addition, a rational drug design approach has been used to identify a peptidomimetic that mimic NS2B/NS3 protease substrate and inhibits its activity. The mechanism proposed for the peptidomimetic is that NS2B/NS3 cleaves between P 1 and P 1 ′ in a peptide substrate consisting of P 2 P 1 P 1 ′, where P 1 and P 2 are basic amino acids (Arg or Lys) and P1′ is a side-chain amino acid (Gly, Ser, or Ala) [ 56 , 231 ] . Thus, a preferred peptide substrate contains several positively charged amino acids. A common method for screening peptide inhibitors of NS2B/NS3 protease employs a fl uorophore conjugated peptide substrate containing basic amino acids at the P1 and P2 positions. Cleavage of peptide substrate by NS2B/NS3 protease results in a release of fl uorophore and increase of fl uorescence [ 242 , 243 ] . Several peptide inhibitors of NS2B/NS3 protease have been identifi ed against WNV [ 62 , 309 -311 ] . A novel agmatine dipeptide inhibitor with improved inhibitory activity against WNV NS2B/ NS3 has been recently identifi ed [ 309 ] . In addition, a recombinant peptide called retrocyclin-1 (RC-1) has been shown to inhibit NS2B/NS3 protease [ 312 ] . However, most of these peptides showed poor activity in the cell-based assay and has not been tested for their in vivo effi cacy. Thus, all of the peptide inhibitor of NS2B/ NS3 protease that has been identifi ed so far failed at the early development stages. Potential limitations of this approach include poor pharmacokinetic properties due to charged nature of peptide, lack of specifi city, requirement of intravenous delivery, rapid degradation in plasma, and costly production.

Dhiraj Acharya and Fengwei Bai RNA interference (RNAi) is a cellular process fi rst described in the nematode Caenorhabditis elegans [ 313 , 314 ] . This process specifically degrades RNA in a sequence-specifi c manner and is conserved in mammalian cells [ 315 , 316 ] . RNAi is a natural defense of eukaryotic cells against viral infections, and may be a promising strategy for developing a potential antiviral therapeutic . Numerous siRNA targets were identifi ed in the genomic region of WNV encoding both structural and non-structural proteins , and siRNA targeting these proteins effectively inhibits WNV replication [ 191 , 317 -321 ] . Besides the siRNA targeting coding regions, siRNA that targets noncoding regions have also been identifi ed to inhibit WNV replication in a sequence-specifi c manner [ 191 ] . Although anti-WNV siRNAs effi ciently block viral replication in cell cultures , similar successes are diffi cult to achieve in animal models [ 191 ] . Quick degradation by serum nucleases, failure to reach target cells, and rapid renal excretion due to their small size and anionic character are hindering the clinical application of antiviral siRNAs. Several delivery systems, including cell-penetrating peptide [ 322 , 323 ] , nanoparticles [ 324 , 325 ] , and viral vectors [ 326 ] , may improve siRNA stability and enhance delivery effi ciency. Despite many challenges , use of antiviral siRNA as anti-WNV therapeutics remains promising.

Cytokines signaling controls diverse immune functions during infection, autoimmune disease, and cancer. Various immunomodulatory or immunostimulatory cytokines and chemokines have been identifi ed to play a protective or pathological role in WNV infection. For examples, type-I interferons (IFNs) [ 327 , 328 ] , interleukin (IL)-23 [ 192 ] , interferon-γ (IFN-γ) [ 83 ] , IL-1β [ 329 ] , macrophage migration inhibitory factor (MIF) [ 330 ] , CXCL10 [ 331 ] , and CCL5 [ 332 , 333 ] protect against WNV infection, whereas IL-10 [ 190 ] and IL-22 [ 334 ] favor WNV pathogenicity. Pharmacological blockade of IL-10 by neutralizing antibody has been shown to protect mice against WNV challenge [ 190 ] . Type I IFNs (IFNα/β) inhibit many fl aviviruses including WNV and have been used as therapeutics against hepatitis C virus [ 335 ] . Although the therapeutic effect of type I IFNs in WNV has yet to be evaluated, its application may be limited due to the antagonistic role of WNV NS5 protein in IFN signaling [ 336 , 337 ] . Interestingly, treatment with pegylated IFN-λ , also known as a type III interferon, has been recently shown to protect mice against lethal WNV infection by decreasing blood-brain barrier permeability [ 338 ] . Thus, strategies targeting the expression of cytokine and chemokine , blocking their signaling, or direct use of recombinant cytokines may be novel approaches for treating WNV infection or controlling its pathology.

Viruses utilize host cellular system for entry, genome replication, transcription , synthesis of viral proteins, and production of viral progenies. In addition, interactions of viral proteins with cellular proteins may evade host immune defense and favor viral replication and pathogenesis . Several host pathways and enzymes including clathrin-mediated endocytosis cyclophilins [ 339 ] , ubiquitin-proteasome system [ 340 ] , unfolded protein response [ 341 ] , nucleotide biosynthesis [ 342 , 343 ] , post-translational protein modifi cation [ 344 -346 ] , and lipid metabolism [ 347 -349 ] have been suggested in fl avivirus replication and pathogenesis. Targeting host factors may be used as a strategy for developing antiviral therapeutic against fl aviviruses, including WNV infection [ 350 -352 ] .

So far, many inhibitors targeting host proteins have been developed and tested against WNV and other fl aviviruses, such as HCV. Host cyclophilin, a family of cellular peptidyl-prolyl isomerases, may serve as a component of fl avivirus replication complex and play a role in fl aviviral replication . Targeting this enzyme by cyclosporine inhibits replication of WNV [ 339 ] . Targeting lipid signaling and metabolism by a bioactive lipid signaling modulator 4-hydroxyphenyl retinamide (4-HPR, fenretinide) also inhibits replication of WNV and other fl aviviruses. Similarly, ribavirin and mycophenolic acid target inosine monophosphate dehydrogenase (IMPDH), an enzyme in purine biosynthesis, and thereby inhibit replication of fl aviviruses [ 342 , 343 ] . In addition, NITD-982 and brequinar that block pyrimidine biosynthesis also inhibit replication of broad range of RNA viruses, including WNV and other fl aviviruses [ 353 , 354 ] . Besides blocking viral genome replication, antiviral targeting of other steps, such as virus maturation, assembly, and viral dissemination into brain has also been suggested and tested against WNV. For instance, inhibitors of alpha-glucosidase I and II, enzymes that play a role in processing of N-linked oligosaccharides of the viral glycoproteins, also inhibit WNV and other fl aviviruses [ 205 , 355 ] . Although the mechanism by which WNV enters the brain is still poorly understood, the two potential routes include axonal retrograde transport (ART) from the peripheral nervous system and direct hematogenous diffusion via a breakdown in the blood-choroid plexus barrier [ 356 ] . Nocodazole, a microtubule inhibitor that blocks ART, delays WNV entry into brain [ 357 ] . The 3′ or the 5′ terminal stem-loop in fl aviviral RNA contains essential cis-acting elements and plays important roles in viral replication [ 358 , 359 ] . Interestingly, a range of cellular proteins have been identifi ed to interact with 3′ stem-loop of fl aviviral RNAs [ 352 , 359 -362 ] , suggesting a potential strategy to design inhibitors targeting this virus-host protein interaction.

Targeting host factors may raise a higher barrier to viral resistance emergence and provide broad-spectrum antiviral effects.

However, current understanding of virus-host cell interaction and research on targeting of host factors to block viral infections are still limited. In addition, there are some potential drawbacks of this approach, including undesirable drug-induced side-effects and diffi culties for drug delivery into brain to control WNV encephalitis . Further understanding of virus-host interaction will facilitate identifi cation of novel antiviral agents.

Development and testing of various methods for treatment and prevention of WNV infection, such as protective vaccines , therapeutic antibodies, antiviral compounds, peptides, and siRNA have been proposed and intensively studied. Although a number of WNV veterinary vaccines have already been licensed and are in use for years, human vaccine candidates are still in various stages of development and testing. Some therapeutic antibodies that show excellent effi cacy in small animal models and are currently being tested in clinical trials represent a promising class of WNV therapeutic. Recent technological advancement and increased understanding of the biology of WNV and other fl aviviruses along with structural/functional characterization of viral proteins have provided a solid foundation for the development of small molecule inhibitors as future WNV therapeutics . However, efforts for development of an effective drug for prevention or control of WNV infection in human still remain unsuccessful. Some of the reasons include a low incidence of diseases, low commercial interest by pharmaceutical companies, high cost of mass vaccination, and diffi culties with running clinical trials due to unpredictable and sporadic nature of WNV outbreaks [ 363 , 364 ] . Another challenge for developing successful WNV therapeutics is to ensure safety and effi cacy in target populations that mostly include children, elderly, and immune-compromised individuals. Despite all these diffi culties, the quest for development of effective treatment and prevention methods against WNV infection are likely to be facilitated by recent technological advancement and should continue to meet the public health needs. 

dengue virus NS5 RNA capping enzyme

West Nile virus methyltransferase domain interacts with protein kinase G

A single amino acid in nonstructural protein NS4B confers virulence to dengue virus in AG129 mice through enhancement of viral RNA synthesis

A single amino acid substitution in the central portion of the West Nile virus NS4B protein confers a highly attenuated phenotype in mice

Inhibition of dengue virus by targeting viral NS4B protein

Chemical genetics strategy identifi es an HCV NS5A inhibitor with a potent clinical effect

B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus

A critical role for induced IgM in the protection against West Nile virus infection

The memory T cell response to West Nile virus in symptomatic humans following natural infection is not infl uenced by age and is dominated by a restricted set of CD8+ T cell epitopes

Comprehensive mapping of West Nile virus (WNV)-and Japanese encephalitis virus serocomplex-specifi c linear B-cell epitopes from WNV non-structural protein 1

Identifi cation of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

Mapping and analysis of West Nile virus-specifi c monoclonal antibodies: prospects for vaccine development

Fusion loop peptide of the West Nile virus envelope protein is essential for pathogenesis and is recognized by a therapeutic cross-reactive human monoclonal antibody

Role of IFN-gamma in an experimental murine model of West Nile virus-induced seizures

Role of CD8+ T cells in control of West Nile virus infection

The relative contribution of antibody and CD8+ T cells to vaccine immunity against West Nile encephalitis virus

Gamma interferon plays a crucial early antiviral role in protection against West Nile virus infection

Pattern recognition receptor MDA5 modulates CD8+ T cell-dependent clearance of West Nile virus from the central nervous system

Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3′-untranslated region

The live attenuated chimeric vaccine rWN/ DEN4Delta30 is well-tolerated and immunogenic in healthy fl avivirus-naive adult volunteers

Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus

An attenuated West Nile prototype virus is highly immunogenic and protects against the deadly NY99 strain: a candidate for live WN vaccine development

Liveattenuated recombinant equine herpesvirus type 1 (EHV-1) induces a neutralizing antibody response against West Nile virus (WNV)

Equine vaccine for West Nile virus

The effi cacy of inactivated West Nile vaccine (WN-VAX) in mice and monkeys

Safety and effi cacy in geese of a PER.C6-based inactivated West Nile virus vaccine

A hydrogen peroxide-inactivated virus vaccine elicits humoral and cellular immunity and protects against lethal West Nile virus infection in aged mice

Development of a new hydrogen peroxidebased vaccine platform

Phylogeny and molecular epidemiology of West Nile and Kunjin viruses

West Nile virus (Kunjin subtype) disease in the northern territory of Australia-a case of encephalitis and review of all reported cases

An inactivated West Nile virus vaccine derived from a chemically synthesized cDNA system

Immunization with West Nile virus envelope domain III protects mice against lethal infection with homologous and heterologous virus

Preparation and immunogenic properties of a recombinant West Nile subunit vaccine

A West Nile virus recombinant protein vaccine that coactivates innate and adaptive immunity

A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice

A novel platform for virus-like particle-display of fl aviviral envelope domain III: induction of Dengue and West Nile virus neutralizing antibodies

Dhiraj Acharya and Fengwei Bai duction of detection and diagnostic reagents of West Nile virus in plants

Plant-made vaccines against West Nile virus are potent, safe, and economically feasible

Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice

The structural immunology of antibody protection against West Nile virus

The molecular basis of antibody-mediated neutralization of West Nile virus

Antibody engineering

Antibody engineering and modifi cation technologies

Glycoengineering of therapeutic antibodies enhances monocyte/macrophage-mediated phagocytosis and cytotoxicity

A paradoxical role for neutrophils in the pathogenesis of West Nile virus

IL-10 signaling blockade controls murine West Nile virus infection

Use of RNA interference to prevent lethal murine West Nile virus infection

Toll-like receptor 7 mitigates lethal West Nile encephalitis via interleukin 23-dependent immune cell infi ltration and homing

Ten years of dengue drug discovery: progress and prospects

Designing screens: how to make your hits a hit

Principles of early drug discovery

Novel trends in high-throughput screening

Review article: high-throughput affi nity-based technologies for small-molecule drug discovery

Fragment screening using X-ray crystallography

Affi nity-based screening techniques: their impact and benefi t to increase the number of high quality leads

Solution structure of dengue virus capsid protein reveals another fold

The fl avivirus precursor membrane-envelope protein complex: structure and maturation

Structure of immature West Nile virus

A high-throughput screening assay for the identifi cation of fl avivirus NS5 capping enzyme GTP-binding inhibitors: implications for antiviral drug development

Discovery of small molecule inhibitors of West Nile virus using a high-throughput sub-genomic replicon screen

HTS identifi es novel and specifi c uncompetitive inhibitors of the two-component NS2B-NS3 proteinase of West Nile virus

In silico screening of small molecule libraries using the dengue virus envelope E protein has identifi ed compounds with antiviral activity against multiple fl aviviruses

Cell-based assays to identify inhibitors of viral disease

Current approaches in antiviral drug discovery against the Flaviviridae family

Novel approaches to fl avivirus drug discovery

Neuroblastoma cell-adapted yellow fever virus: mutagenesis of the E protein locus involved in persistent infection and its effects on virus penetration and spread

The interactions of the fl avivirus envelope proteins: implications for virus entry and release

Infectious entry of West Nile virus occurs through a clathrinmediated endocytic pathway

HIV-1 entry inhibitors: an overview

Targeting HIV-1 gp41-induced fusion and pathogenesis for anti-viral therapy

A ligand-binding pocket in the dengue virus envelope glycoprotein

Structure of a fl avivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

Design, synthesis, and biological evaluation of antiviral agents targeting fl avivirus envelope proteins

A small molecule fusion inhibitor of dengue virus

A small-molecule dengue virus entry inhibitor

Antiviral compounds discovered by virtual screening of small-molecule libraries against dengue virus E protein

Sulfated Escherichia coli K5 polysaccharide derivatives inhibit dengue virus infection of human microvascular endothelial cells by interacting with the viral envelope protein E domain III

Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry

Antiviral effect of the heparan sulfate mimetic, PI-88, against dengue and encephalitic fl aviviruses

Antiviral activity against dengue virus of diverse classes of algal sulfated polysaccharides

Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?

Antiretroviral treatment of adult HIV infection: 2014 recommendations of the International Antiviral Society-USA Panel

Protease inhibitors as antiviral agents

Discovery and development of VX-950, a novel, covalent, and reversible inhibitor of hepatitis C virus NS3.4A serine protease

Preclinical profi le of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins

NMR analysis of a novel enzymatically active unlinked dengue NS2B-NS3 protease complex

High-throughput screening of low molecular weight NS3-NS4A protease inhibitors using a fl uorescence resonance energy transfer substrate

Establishment of an in vitro assay system for screening hepatitis C virus protease inhibitors using high performance liquid chromatography

A novel high throughput screening assay for HCV NS3 serine protease inhibitors

Non-peptide inhibitors of HCV serine proteinase

The fl avivirus protease as a target for drug discovery

Structural evidence for regulation and specifi city of fl aviviral proteases and evolution of the Flaviviridae fold

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Investigators of The Multicenter Study of Perioperative Ischemia Research Group, Ischemia Research, and Education Foundation (2007) Mortality associated with aprotinin during 5 years following coronary artery bypass graft surgery

Discovery of a non-peptidic inhibitor of West Nile virus NS3 protease by high-throughput docking

Identifi cation and biochemical characterization of small-molecule inhibitors of West Nile virus serine protease by a high-throughput screen

Characterization of the West Nile virus protease substrate specifi city and inhibitors

Synthesis and in vitro evaluation of West Nile virus protease inhibitors based on the 2-{6

Structure-activity relationship and improved hydrolytic stability of pyrazole derivatives that are allosteric inhibitors of West Nile Virus NS2B-NS3 proteinase

Inhibitors of Dengue virus and West Nile virus proteases based on the aminobenzamide scaffold

Synthesis and in vitro evaluation of West Nile virus protease inhibitors based on the 1,3,4,5-tetrasubstituted 1H-pyrrol-2(5H)-one scaffold

Design, synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases

Small molecule pan-dengue and West Nile virus NS3 protease inhibitors

Flexibility between the protease and helicase domains of the dengue virus NS3 protein conferred by the linker region and its functional implications

Structure of West Nile virus NS3 protease: ligand stabilization of the catalytic conformation

A single positively selected West Nile viral mutation confers increased virogenesis in American crows

Viral determinants in the NS3 helicase and 2 K peptide that promote West Nile virus resistance to antiviral action of 2′,5′-oligoadenylate synthetase 1b

Purifi cation and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme

Direct fl uorometric measurement of hepatitis C virus helicase activity

Identifi cation and analysis of inhibitors targeting the hepatitis C virus NS3 helicase

Identifi cation and analysis of hepatitis C virus NS3 helicase inhibitors using nucleic acid binding assays

High-throughput screening assay of hepatitis C virus helicase inhibitors using fl uorescence-quenching phenomenon

Viral and cellular RNA helicases as antiviral targets

Halogenated benzimidazoles and benzotriazoles as inhibitors of the NTPase/ helicase activities of hepatitis C and related viruses

Synthesis and evaluation of ATP-binding site directed potential inhibitors of nucleoside triphosphatases/helicases and polymerases of hepatitis C and other selected Flaviviridae viruses

An analogue of AICAR with dual inhibitory activity against WNV and HCV NTPase/helicase: synthesis and in vitro screening of 4-carbamoyl-5-(4,6-diamino-2,5-dihydro-1,3,5-triazin-2-yl)imidazole-1-beta-D-ribo furanoside

Ring-expanded ("fat") nucleoside and nucleotide analogues exhibit potent in vitro activity against fl aviviridae NTPases/helicases, including those of the West Nile virus, hepatitis C virus, and Japanese encephalitis virus

Potent inhibition of NTPase/helicase of the West Nile Virus by ring-expanded ("fat") nucleoside analogues

Characterization of imidazo[4,5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus nucleoside triphosphatase/helicase: evidence for activity on the level of substrate and/ or enzyme

Ivermectin is a potent inhibitor of fl avivirus replication specifi cally targeting NS3 helicase activity: new prospects for an old drug

New insights into fl avivirus nonstructural protein 5

Comparative mechanistic studies of de novo RNA synthesis by fl avivirus RNA-dependent RNA polymerases

The fl avivirus polymerase as a target for drug discovery

Advances in the development of new therapeutic agents targeting the NS3-4A serine protease or the NS5B RNA-dependent RNA polymerase of the hepatitis C virus

Recent highlights in the development of new antiviral drugs

Identifi cation of a small molecule that inhibits herpes simplex virus DNA polymerase subunit interactions and viral replication

A homogeneous, solid-phase assay for hepatitis C virus RNA-dependent RNA polymerase

A cell-based reporter assay for inhibitor screening of hepatitis C virus RNA-dependent RNA polymerase

A continuous nonradioactive assay for RNA-dependent RNA polymerase activity

Synergistic combinations of favipiravir and oseltamivir against wild-type pandemic and oseltamivir-resistant infl uenza A virus infections in mice

In vitro activity of favipiravir and neuraminidase inhibitor combinations against oseltamivir-sensitive and oseltamivir-resistant pandemic infl uenza A (H1N1) virus

Favipiravir (T-705), a novel viral RNA polymerase inhibitor

Broadspectrum inhibitor of viruses in the Flaviviridae family

A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties

A dengue fever viremia model in mice shows reduction in viral replication and suppression of the infl ammatory response after treatment with antiviral drugs

Crystal structure of the dengue virus RNAdependent RNA polymerase catalytic domain at 1.85-angstrom resolution

Inhibition of dengue virus polymerase by blocking of the RNA tunnel

Conformational fl exibility of the dengue virus RNA-dependent RNA polymerase revealed by a complex with an inhibitor

An RNA cap (nucleoside-2′-O-)-methyltransferase in the fl avivirus RNA polymerase NS5: crystal structure and functional characterization

Structure and function of fl avivirus NS5 methyltransferase

2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members

2′-O methylation of internal adenosine by fl avivirus NS5 methyltransferase

Structural and functional analyses of a conserved hydrophobic pocket of fl avivirus methyltransferase

A structural basis for the inhibition of the NS5 dengue virus mRNA 2′-O-methyltransferase domain by ribavirin 5′-triphosphate

Flaviviral methyltransferase/RNA interaction: structural basis for enzyme inhibition

Biochemical characterization of the (nucleoside-2′O)-methyltransferase activity of dengue virus protein NS5 using purifi ed capped RNA oligonucleotides (7Me) GpppAC(n) and GpppAC(n)

Flavivirus methyltransferase: a novel antiviral target

The fl avivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a twostep reaction to form the RNA cap structure

Structure-based and ligand-based virtual screening of novel methyltransferase inhibitors of the dengue virus

Small molecule inhibitors that selectively block dengue virus methyltransferase

Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs

Identifi cation of a novel antiviral inhibitor of the fl avivirus guanylyltransferase enzyme

Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap

Novel inhibitors of dengue virus methyltransferase: discovery by in vitro-driven virtual screening on a desktop computer grid

5′-Silylated 3′-1,2,3-triazolyl thymidine analogues as inhibitors of West Nile virus and dengue virus

Phage display of combinatorial peptide libraries: application to antiviral research

Design and structure of peptide and peptidomimetic antagonists of protein-protein interaction

HIV-1 gp41 as a target for viral entry inhibition

Antiviral peptides targeting the West Nile virus envelope protein

Peptide inhibitors of dengue virus and West Nile virus infectivity

Novel agmatine dipeptide inhibitors against the West Nile virus NS2B/NS3 protease: a P3 and N-cap optimization study

Tripeptide inhibitors of dengue and West Nile virus NS2B-NS3 protease

Peptide inhibitors of West Nile NS3 protease: SAR study of tetrapeptide aldehyde inhibitors

Inhibition of dengue NS2B-NS3 protease and viral replication in Vero cells by recombinant retrocyclin-1

Potent and specifi c genetic interference by double-stranded RNA in Caenorhabditis elegans

RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans

Sequence-specifi c inhibition of microRNA-and siRNA-induced RNA silencing

Mechanisms of gene silencing by double-stranded RNA

Actively replicating West Nile virus is resistant to cytoplasmic delivery of siRNA

A single siRNA suppresses fatal encephalitis induced by two different fl aviviruses

The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negativeand positive-strand RNA viruses

Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication

Inhibition of West Nile virus replication in cells stably transfected with vector-based shRNA expression system

Transvascular delivery of small interfering RNA to the central nervous system

RVGpeptide-linked trimethylated chitosan for delivery of siRNA to the brain

Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

Delivery of multiple siRNAs using lipid-coated PLGA nanoparticles for treatment of prostate cancer

Inhibition of West Nile virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells

Alpha/ beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival

Beta interferon controls West Nile virus infection and pathogenesis in mice

IL-1beta signaling promotes CNS-intrinsic immune control of West Nile virus infection

Abrogation of macrophage migration inhibitory factor decreases West Nile virus lethality by limiting viral neuroinvasion

T-cell recruitment and control of West Nile virus encephalitis

A role for the chemokine RANTES in regulating CD8 T cell responses during chronic viral infection

Chemokine receptor CCR5 promotes leukocyte traffi cking to the brain and survival in West Nile virus infection

IL-22 signaling contributes to West Nile encephalitis pathogenesis

Clinical-and cost-effectiveness of pegylated interferon alfa in the treatment of chronic hepatitis C: a systematic review and economic evaluation

The NS5 protein of the virulent West Nile virus NY99 strain is a potent antagonist of type I interferon-mediated JAK-STAT signaling

Flavivirus antagonism of type I interferon signaling reveals prolidase as a regulator of IFNAR1 surface expression

Interferon-lambda restricts West Nile virus neuroinvasion by tightening the blood-brain barrier

Cyclosporine inhibits fl avivirus replication through blocking the interaction between host cyclophilins and viral NS5 protein

The ubiquitin-proteasome pathway is important for dengue virus infection in primary human endothelial cells

Dengue virus serotype infection specifi es the activation of the unfolded protein response

Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA

The predominant mechanism by which ribavirin exerts its antiviral activity in vitro against fl aviviruses and paramyxoviruses is mediated by inhibition of IMP dehydrogenase

Increased pathogenicity of West Nile virus (WNV) by glycosylation of envelope protein and seroprevalence of WNV in wild birds in Far Eastern Russia

Glycosylation of the West Nile virus envelope protein increases in vivo and in vitro viral multiplication in birds

Multiple amino acid changes at the fi rst glycosylation motif in NS1 protein of West Nile virus are necessary for complete attenuation for mouse neuroinvasiveness

U18666A, an intra-cellular cholesterol transport inhibitor, inhibits dengue virus entry and replication

West Nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids

West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin

Dhiraj Acharya and Fengwei Bai Discovery of insect and human dengue virus host factors

Exploitation of cellular pathways by dengue virus

Host factors involved in West Nile virus replication

Characterization of dengue virus resistance to brequinar in cell culture

Inhibition of dengue virus through suppression of host pyrimidine biosynthesis

Novel imino sugar derivatives demonstrate potent antiviral activity against fl aviviruses

Mechanism of West Nile virus neuroinvasion: a critical appraisal

Nocodazole delays viral entry into the brain following footpad inoculation with West Nile virus in mice

Identifi cation of cis-acting nucleotides and a structural feature in West Nile virus 3′-terminus RNA that facilitate viral minus strand RNA synthesis

Cell proteins TIA-1 and TIAR interact with the 3′ stem-loop of the West Nile virus complementary minus-strand RNA and facilitate virus replication

BHK cell proteins that bind to the 3′ stem-loop structure of the West Nile virus genome RNA

Interaction between the cellular protein eEF1A and the 3′-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis

Cell proteins bind specifi cally to West Nile virus minus-strand 3′ stem-loop RNA

Cost-effectiveness of West Nile virus vaccination

Prospects for development of a vaccine against the West Nile virus

Immune responses to an attenuated West Nile virus NS4B-P38G mutant strain

Phase II, randomized, double-blind, placebo-controlled, multicenter study to investigate the immunogenicity and safety of a West Nile virus vaccine in healthy adults

The authors are very thankful to Dr. Dobrivoje S. Stokic for his critical reading. This work is supported by funding from Wilson Research Foundation, Jackson, MS, NIH R15AI113706, and the University of Southern Mississippi.