Flaviviruses are neurotropic, but how do they invade the CNS?

YJINF3324_proof ■ 3 June 2014 ■ 1/13 Journal of Infection (2014) xx, 1e13

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www.elsevierhealth.com/journals/jinf

REVIEW

Flaviviruses are neurotropic, but how do they invade the CNS? Q7

J.W. Neal Institute of Infection and Immunity, Henry Wellcome Building, University Hospital of Wales, Cardiff University, Cardiff CF14 4XN, United Kingdom Accepted 19 May 2014 Available online - - -

KEYWORDS Flavivirus; Brain; Inflammation; Axonal transport; Blood brain barrier; Tight junction proteins

Summary Flaviruses (FV) are RNA viruses carried by mosquitoes. Neurological signs including acute encephalitis, meningitis and acute flaccid paralysis develop in a small percentage of infected individuals; long term sequlae are, Parkinsonism, dystonias and cognitive changes. FV neuroinfection is neurotropic involving subcortical nuclei (substantia nigra and thalamus) anterior horn neurons and neocortex. Glycosylation of the FV E envelope protein is one determinant of neuroinvasion, increasing both axonal and trans-epithelial transportation. Neutralizing antibodies against the E and NS proteins prevents FV uptake into several cell types, including axons. CD8þ T cells are vital for clearance of WNF infected cells from the CNS, whereas TLR-3 and TLR-7 mediated anti-virus response through increased serum inflammatory cytokines to disrupt the BBB providing infected leucocytes and free virus access to the CNS (so called Trojan horse) Cellular virus attachment factors, promoting FV cell entry are widely distributed and include DC-SIGN (that detects complex carbohydrate molecules); Glycosamino glycans (GAG), Heparan sulphate(HSPG) Semaphorin 7A, Low Density Lipid receptors (LDLR); these are not FV specific virus entry receptors. The FV also crosses epithelial and endothelial barriers by disrupting Tight Junction complexes to increase BBB permeability. This review describes the multiple pathways responsible for the neuroinvasive properties of the Flaviviruses. ª 2014 Published by Elsevier Ltd on behalf of The British Infection Association.

Introduction West Nile Fever Virus (WNF), Japanese Encephalitis Virus (JEV), Dengue Virus (DV), Murray Valley Encephalitis Virus (MVE), St Louis encephalitis Virus (SLEV) and Hepatitis C

(HCV) are members of the Flaviviridae family (FV). FV are enveloped, positive sense, single stranded RNA viruses (ssRNA) with significant neuroinvasive characteristics and are regarded as neurotropic viruses. Horses, pigs, birds (Corvid species) and dogs all provide a natural reservoir

E-mail address: [email protected] http://dx.doi.org/10.1016/j.jinf.2014.05.010 0163-4453/ª 2014 Published by Elsevier Ltd on behalf of The British Infection Association. Please cite this article in press as: Neal JW, Flaviviruses are neurotropic, but how do they invade the CNS?, J Infect (2014), http:// dx.doi.org/10.1016/j.jinf.2014.05.010

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Q2

for the FV.1e4 Whereas, humans are regarded as “dead end hosts” because they are infected accidentally when bitten by a Flavivirus (FV) carrying mosquito, usually from the Culex species (WNF, JEV)3,4; the Aedes aegypti and albopictus mosquitoes are responsible for DV infection.5 Of all the FV, WNF and JEV, are the two most commonly associated with neuroinfection. WNF is classified as linage I and 2 strains; strains I (North America) are neuro virulent, whereas, linage 2 are less virulent and found in Sub Saharan Africa. WNF infection is mainly asymptomatic, but in approximately 20% of cases there is a flu-like illness with pyrexia and joint pains. However, a small percentage of WNF infected individuals, (1 in 150)2e4,6 develop meningoencephalitis 14 days after infection and in some an acute flaccid paralysis (AFP).7,8 For JEV, the ratio of symptomatic to asymptomatic infection is varies between 1 in 25 to 1in 1000 cases, neuro infection is more frequent than WNF, with up to 60% individuals experiencing a seizure as well as AFP.10e13 Comparable data regarding the frequency of neuroinfection in MEV and SLEV is not available, but DV infection (one of four serotypes DNEV1-4) is more likely to result in haemorrhagic shock than primary neuroinfection.14e16 Recent reports have described extrahepatic neuroinfection with both DV and HCV, but neuropathological information is limited.17,18 Despite the evidence for FV being neurotropic little is known about the factors responsible for neuroinvasion.9,10,19,20 Clinical and neuropathological findings, show the FV cross the blood brain barrier (BBB) and choroid plexus (CPLx)together with evidence for axonal transport from the periphery (skin bite) and through olfactory bulb neurons(systemic viremia) into the CNS.21,22 The identification the virus entry receptors and the anti-virus intra cellular signalling pathways related to FV neuroinvasion will help to define potential therapeutic targets.19 This review will describe the clinical, neuropathological and experimental information related to current understanding of neuroinvasion by the FV (Table 1).

The clinical consequences of Flaviviruses neuroinfection WNF and JEV are responsible for acute encephalitis and meningitis are with cognitive impairment23e27; movement disorders such as AFP,28 dystonia, Parkinsonism are reported in both WNF and JEV.29e34 Initially, AFP was regarded clinically as Guillian-Barre syndrome (GBS) and electrophysiological examination was consistent with demyelination and involvement of anterior spinal horn cells. Autopsy examination confirmed these findings, but found anterior spinal horn neuronal loss.30e35 Therefore, AFP is regarded as polio-like lower motor neuron disorder affecting the lower limbs, this has also been reported in MVF and SEV.36e40 The involvement of the autonomic nervous system is common with FV infection with disorders of cardiac conduction and sphincter control.33,34 Neurological involvement with DV is rare (6%) but includes quadriplegia, transverse meylitis, GBS syndrome and encephalopathy.14,15,41 For HCV, about 6%of cases have cognitive impairment, vascular changes relating to endothelial infection, demyelination and inflammatory white

J.W. Neal matter changes. In up to 50% ofHCV infected individuals there is evidence of circulating cryoglobulins (cold deposited immunoglobulins) (CG) capable of causing vascular injury and a peripheral neuropathy17,42e45; there is some evidence the level of circulating CG corresponds to the level of impaired cognitive function.44 Neuroradiological findings in JEV, WNF, MEV and SLEV all show an increased MRI T2 weighted signal from the thalamus, brain stem and basal ganglia.38,39,45,46 MRI signal changes in the anterior horn spinal cord are consistent with AFP in WNF, MEV and JEV.2,23,46e50 Dystonias and Parkinsonism in SLEV, WNF and JEV was correlated with loss of neurons from the brain stem nuclei from each of these virus infections.46e48 For HCV, the white matter and periventricular changes are attributed to small vessel disease and demyelination.17 Neuropathology of the FV demonstrates encephalitis and meningitis, but more specifically infection of neurons in the substantia nigra nucleus (SN), thalamus, cerebellum and cerebral cortex,9,55 these findings mirror the neuroradiological findings and explain Parkinsonism, dystonias and AFP.28 Immunohistochemical (ICH) evidence found WNF (Envelope and non-structural NS1 proteins) within neurons and glia.56 In WNF infected astrocytes from autopsy tissue contained persistent virus infection for up to 114 days, providing an explanation for the persistence of neurological signs.54 JEV virus antigen positive neurons and astrocytes are present in thalamus, hippocampus, SN and brain stem49e52 whereas in vitro, JEV infects neurons, astrocytes and microglial.57 The neuropathology of HCV includes perivascular T cells and microglial nodules. ICH staining of HCV autopsy tissue found infected HVC in microglia and astrocytes, but unlike the other FV infections, neurons were not immunolabelled, however negative strand HCV RNA, was detected suggestive of HCV replicating within the CNS.1,18 HCV infected microglia produce a range of proinflammatory cytokines Tumour necrosis factor (TNF) and interleukins (IL-1, IL-12 and IL18) all capable of promoting tissue injury and altering BBB integrity.42e45 The presence of inflammatory cytokines would also correspond to episodes of de meylination and recurrent transverse meylitis.17,44

The host immune response to Flavi virus infection and the risk of neuroinvasion The host’s innate and adaptive immune system response to systemic FV infection is vital in order to prevent neuroinvasion, a factor emphasized by increased risk of WNF in individuals with a compromised immune system. Although the risk of neuroinfection with systemic WNF infection is small, it is increased with age (20 fold increase over 50 years of age), male sex, diabetes mellitus, hypertension, systemic malignancies, trans fusion of WNF infected blood products and transplant recipients receiving organs from infected WNF donors.6e8,20e22 WNF infection is uniformly fatal in Severe combined immunodeficiency involving both T and B cells and in humans with various immuno deficiency syndromes.59,60 Similarly, for JEV, an intact immune system prevents neuroinvasion in both animal and human cases, but unlike WNF, neuroinfection with JEV declines in adolescents.11

Please cite this article in press as: Neal JW, Flaviviruses are neurotropic, but how do they invade the CNS?, J Infect (2014), http:// dx.doi.org/10.1016/j.jinf.2014.05.010

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Table 1 The table shows the potential virus entry receptors related to FV neuroinfection and their CNS distribution. The CNS contains neurons and glia together with perivascular endothelial cells also express receptors .The blood brain barrier components include astrocytes and perivascular cells also express FV receptors. The distribution of potential virus entry receptors is correlated with their cellular distribution. The function of the potential virus entry receptors id given together with the putative ligand on the individual FV viruses. Potential FV entry receptor

CNS cell expressing entry receptors

Virus entry detection receptor function

Flavivirus ligand detected

MMR (CD206) C-Type lectin

Meninges Perivascular macrophages Astrocytes

DV E protein

DCSIGN(CD209) Dendritic cells specific intercellular-3 grabbing non integrin. C-Type lectin

Meninges Perivascular macrophages Endothelial cells

Mannose carbohydrates on viruses endocytosed and processed for antigen presentation Mannose complex internal branched glycoproteins on virus endocytosed for antigen processing

TRL 3 TRL-7

Ependyma Neurons, Microglia astrocytes Peripheral lymphocytes myeloid cells Microglia, astrocytes Skin Dendritic cells Neurons in vitro Microglia, astrocytes

Detect WNF ds RNA to initiate anti virus response and inflammatory cytokines Detects WNF dsRNA viaMyD88 increases IL-23 promoting macrophages into CNS Detects short ssRNA

Fibroblasts endothelial and epithelial cells Astrocytes Microglia Neurons Glia Inter endothelial cells and potentially between CPLX Neurons

Extracellular loop of CD81 binds to E in HCV

RIG-1

CD81 Tetraspandin

Claudins-1, -6, -9

Low density lipoprotein receptor (LDLR) Glycoaminoglycans (GAG)

Heparan sulphate proteoglycans (HSPG) Heat Shock Proteins (hsp)

Wide cellular distribution

Wide cellular distribution Neuron in vitro

The host’s adaptive system immune response to FV infection and clearance from the CNS thi Systemic CD8þ T cell lymphocytes are vital for defence against WNF because they recognize WNF infected cells expressing MHC class I molecules and WNF proteins containing multiple peptide regions.16 The peptide sequences from WNF M (membrane protein), E (envelope protein), NS3 and NS4 (Non-structural proteins) elicit the most frequent response from CD8þ T cells.61 CD8þ T are responsible for lysing infected cells through production of pro inflammatory cytokines such as interferon (IFN), granz enzymes A and B, perforins.62,63 CD8þ T cells are also

Tight junction proteins WNF capsid protein promotes claudin microtubule transport to lysosomes and degradation LDLR detects ApoE increases endocytosis GAGs similar to complex carbohydrates on viral proteins

HSPG similar to carbohydrate complexes on virus proteins Interact with TRL-7 to promote microglial clearance of WNF

Complex carbohydrates on WNF JEV DV E protein N linked glycans HCV WNF ds RNA DV ss RNA

WNFssRNA JEV ssRNA HCV DV HCV E1 and E2 proteins

WNF capsid protein, JEV, HCV DVprM/Minteracts with claudin-1 HCV Apoli protein within the E protein WNF JEV domain III of E protein MEV domain I of E protein DV domain III of E DV E protein HCV DV JEV

responsible for initiating apoptosis of infected WNF cells through the FaseFas ligand,CD40-CD 40 ligand64 and Tumour necrosis factor related-apoptosis-inducing ligand (TRAIL),65 this promotes their clearance and prevents persistent CNS WNF infection .The importance of CD8þ T cells is supported by higher viral loads in either CD8þ T or IFN mice deficient after systemic WNF infection.66 The presence of adequate numbers of CDT8þ cells within the brain are vital in order to clear WNF infected neurons and glia.16,67 The production of systemic cytokines TNF and IFN increases the trafficking of infected CD8þ T cells,CD45þ leucocytes and CD11bþ cells into the CNS.66,67 whereas, chemokines (CCL5 and RANTES)


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expressed by infected glia and neurons (CXCL10) increases trafficking into the CNS of virus specific CD8Tþ cells expressing the CXR3 receptor.68 Neuroinvasion begins in the caudal parts of the CNS and proceeds rostrally, this corresponds to the selective up regulation of the CXCL10 receptor, CXCR3, by cerebellar neurons increasing local trafficking of WNF CD8þ T cells.68,69 Deficiency of CXCL10, or its receptor CXCR3, reduces CD8þ T cell entry into the CNS, specifically the cerebellum, preventing virus clearance and increasing CNS virus load.65 A further chemokine, CXCL12, expressed by the BBB also regulates neutrophil entry into the CNS promoting the clearance of WNF and reducing mortality.71 Conversely, a deficiency in the cytokine receptor, CCR5, in mice increases virus load, as it reduced neutrophil influx in to the CNS and a polymorphism of the CCR5 gene is associated with an increased risk of WNF neuroinfection in human.72,73

CD4D T maintain protective Ig levels and FV clearance from the CNS CD4þ T cells have multiple functions in response to WNF infection producing IFN and IL-2,a direct anti virus effect and priming B cells helping to sustain anti-WNF IgG levels during the later stages of WNF infection.74 By maintaining IgG levels CD4þ T cells promote trafficking of CD8þ T cells into the CNS contributing to the effective clearance of WNF infected cells from the CNS.74 B cell deficient mice that are unable to produce either anti-WNF IgM or IgG antibodies develop an encephalitis, but they are protected by passive transfer of IgG before WNF infection confirming that host protection against FV infection requires Ig related effects such as opsonization and complement activation.75e78 WNF infecting DC (Dendritic cells) within lymphoid tissue initiates anti-WNF Ig production with IgM levels coinciding with systemic virus clearance, whereas, WNF specific IgG is produced later, only after CNS infection has become established75e77; therefore, despite the evidence supporting IgG promoting CD8þ T cell trafficking into the CNS its the role in relation to WNF neuro infection is not clear.16,78

Complement pathway activation in response to FV infection Glia and neurons are capable of expressing the full range of complement (C) pathway components. WNF and WNFantibodies activate the classical pathway (C), this contributes to the anti-viral response through opsonisation of infected cells with, C3b and C4b this facilitates the clearance of infected cells by phagocytic Fc receptors, cell lysis through formation of the C5eC9 complex and chemotaxis of microglia into the infected CNS.79e81 Mice deficient in various components of the C classical and alternative pathways (C1q, CR1 and C3) are vulnerable to WNF infection, because this virus has multiple effects upon of the innate immune response, including the reduced opsonisation of WNF infected cells with C3 and C4 and prevention of protective anti-WNF antibody (IgG or IgM) binding to Fc phagocytic receptors82e84 WNF non-structural proteins

J.W. Neal also inhibit C activation, preventing the clearance of WNF from the CNS.82

Neutralizing anti bodies against E and non structural proteins potential therapy Virus entry to host cells involves three stages; attachment, internalization and fusion with host cell membrane. This process requires caltherin, early and late endosomal compartments (Rab-5þ, Rab 7) mediated endocytosis and low pH related conformational changes on FV proteins that drive membrane fusion.85e87 During infection many neutralizing antibodies are directed against one of the three domains I, II and III of the E structural protein and because they inhibit FV attachment to host cells have therapeutic applications.85e88 A humanized Mab (E16) against E protein, domain III, provided the most protection, because it inhibited the conformational changes in E protein at the post attachment stage blocking early virus entry. When Mab (E16) was administered to WNF infected mice it crossed the BBB and stimulated virus clearance from the CNS and from infected neurons.86e88 A recombinant human anti-body targeting E protein (MGAWN1) has in a phase 1 trial produced neutralizing antibodies, crossed the BBB and was safe, confirming neutralizing antibodies against FV represent potential therapeutic agents.89,90 Neutralizing antibodies against WNF non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) also increased the clearance of WNF virus by opsonizing targets for Fc receptors, activation of complement and increased phagocytosis of IgG immune complexes by macrophages and microglia.91,92

Molecular characteristics of E protein are related to the risk of neuroinfection The increased risk of encephalitis in WNF epidemics is associated with E protein glycosylation. This property has been confirmed in mice inoculated with the highly neuro virulent WNF New York (NY) 99strain (E protein glycosylated), whereas the non-glycosylated WNF virus strain (ETH 76) did not produce neuroinvasion. Neuroinvasive properties are not restricted to E because changes in amino acids in NS1 reduced neuroinvasion.93e96 Similar studies with JEV found amino acid changes in the E protein determined neuroinvasion in mice, but it is not clear if these E protein changes are responsible for a higher frequency of neuroinvasion in humans after systemic infection in JEV as compared to WNF.97 For both MEV and SLVE, glycosylation of E protein motif in domain I also increases stability of the protein in acidic pH, this increases the neuroinvasive properties as found after inoculation of the NY 99 strain of WNF in mice.98,99 Changes in an amino acid in the hinge region between Domains I and II increasing stability of the virus and increasing its capacity to fuse with host cells.98,99 The E protein in DV has two N linked glycans at Asn 153 (highly conserved in FV) and Asn-67 (specific for DV), the later is linked to attachment to host receptors (Dendritic Cell-Specific Intercellular molecule-3 grabbing Non-integrin (-CD209) DC-SIGN and is vital for virus propagation100,101; removal of glycan 67 reduced DV infection of on macrophages and dendritic


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cells.103,104 However, these changes are not associated with increasing the risk of DV neuroinvasion, because DC-SIGN provides attachment rather than virus entry. For HCV, the N ectodomains of E 1and E2 glyco proteins are responsible for virus attachment; they are glycosylated and bind to the extra cellular loop of CD81, although this has not been identified in brain cells.102

in vitro promoted a pro inflammatory, anti-viral response, through RIG-1 dependent pathways and inhibited neuroinvasion.112 Deficiency of RIG-1 and MDA-5 in mice infected with WNF increased virus replication and neuroinvasion, confirming the importance of RIG-1 for initiating the IFNprotective response expressed by neurons and glia against JEV and WNF.111e114

Genetic factors influencing the regulation of the immune response increases the risk of flavivirus neuro infection

TRL detection of FV; a balancing act with a double edged sword

The risk of WNF increases in individuals with Single Nucleotide Polymorphism (SNP) in several genes responsible for immune regulation and anti-viral response.72,73,105,106 The CCR5 delta 32 polymorphisim in the gene for the chemokine receptor CCR5 is present in about 1%of the United States population and is a potential risk factor for WNF neuroinfection, although this is not without controversy.72,73,107 Mice with Cccr5 gene deficiency show increased virus load after WNF infection and human CCR5 deficiency is a risk factor for neurological sequel after WNF infection,72,73,107 a factor attributed to reduced neutrophil influx in to the CNS associated with increased virus load. SNPs in the IRF3 and MX1,105 both immuno regulatory genes inhibit host IFN response to WNF as do SNPs in oligoadenylate synthetase 1(OAS1)105 also an interferon regulatory gene and non-sense mutation in an anti viral enzyme 20 ,5-oligoadenylate synthetase genes increased risk and severity of WNR neuroinfection in humans and horses respectively.108 Interestingly a polymorphism in DC-SIGN on macrophages and dendritic cells reduced FV attachment to cell membranes.85,103

Host cells express PRR and complex carbohydrate molecules (cell attachment factors) to detect and inhibit FV neuroinvasion Most WNF infections are asymptomatic and do not result in either a systemic illness or neuroinfecton, so the host immune system, including the CNS must have an effective antiviral response .In the CNS neurons, glia, CPLX and the BBB endothelium are all part of the innate immune response and pattern recognition receptors (PRR) that detect non-host, unique, pathogen associated molecular patterns (PAMPs) including viral nucleic acids and complex glycoproteins, for example the FV structural and nonstructural proteins.109,110 PRR include Toll-like receptors (TLR), RIG-1 (retinoic acid inducible gene-1 short RNA) (RIG-1), melanoma differentiation antigen-5 (MDA-5) long RNA.111e113 Whereas, the Mannose membrane receptor (MMR) and DC-SIGN function as PRR because of their C-terminal carbohydrate recognition domain (CRD) that detects complex, non-host, carbohydrate molecules, as found on many viruses.103,104,109,110 WNFV, ssRNA, is detected by the intra-cytoplasmic RIG-1 and MDA5, these PRR are both expressed by tissue macrophages, microglia and astrocytes: RIG-1 also detects JEV and HCV nucleic acids.109,111,112 PRR detection of viral nucleic acids initiates various intracellular signalling pathways of the protective host anti-viral IFN type I response.113,114 JEV infection of neurons

TLR 3 is expressed on either the cell surface or endosomal compartment by a wide range of host cells, including neurons and glia; TLR3 detects WNF ds RNA inside cells and initiates intracellular signalling pathways to promote an anti-virus IFNresponse.109 In WNF infected mice, deficient for TLR-3, demonstrated a reduced serum IL-6, IFN and TNF levels, but the mice were in fact more resistant to WNF neuroinvasion with better survival, fewer infiltrating leukocytes and less virus inside the brain.115 This was explained by less BBB permeability in TLR3/ mice due to reduced serum inflammatory cytokine levels (especially TNF) and supporting the interpretation that TLR3 signalling was infact a “double edged sword” not only responsible for the anti-viral response, but also potentially increasing WNF neuroinfection.115 However, a similar study using TLR3 deficient mice, infected with WNF, found TLR3 had a predominatey protective role and did not increase susceptibility to neuroinfection.116 The innate immune system also responds to WNF in many ways independently of TLR3 expression; macrophage inhibitory factor (MIF) is increased in CNS virus infection, but in Mif/ deficient mice WNF inoculation produced increased clinical survival and reduced neuroinvasion due to lower TNF levels, a finding independent of TLR3 expression117,118; WNF NS-1protein inhibits TRL3 signalling preventing IFN production119; WNF RNA is masked preventing its detection and promoting persistent glial and neuronal infection111 and the interaction between C type lectins (DC SIGN) with pathogens also inhibits TLR related cytokine signalling promoting FV local neuroinvasion.103 In macrophages infected with WNF glycosylated E protein prevented viral ds RNAinduced proinflammatory cytokine production by blocking distal sites where multiple intracellular cytokine signalling pathways converge.120 TLR 7detects ssRNA expressed by infected Langerhans skin cells (similar to dendritic cells), neurons and microglia; it is a cytoplasmic receptor for uridine and guanosine rich ssRNA WNF. In wild type animals with WNF encephalitis the presence of TLR 7 increased the expression of microglial interlekinIL-23, increasing the influx into the CNS of CD11Bþ macrophages and CD45þ leucocytes resulting in neutralization and clearance of the WNFvirus infected cells121;TRL-7 also cooperates with Heat-shock protein 70 to promote macrophage phagocytizes of WNF infected cells. WNF neuroinfection is increase in TLR 7deficient animals because microglia and leucocytes were unable express IL-23 and detect infected WNF cells .On this basis, systemic TLR7 signalling through IL-23 to reduces WNF neuroinvasion and its role signalling immune cells into the WNF infected CNS is consistent with the function of CCR5, a chemokine


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receptor also important for leucocyte trafficking into WNF infected CNS; a deficiency of this chemokine also results in an increased risk of WNF neuroinvasion.107 Similarly, a deficiency of an adaptor molecule myeloid differentiation factor 88 (MyD 88) a vital component for TRL-7 related anti-virus proinflammatory cytokine expression is associated with increased WNF replication inside neurons.120,121

Trans-membrane receptors MMR and DCSIGN; potential virus entry receptors for FV neuroinvasion? MMR, detects single mannose branched structures, whereas, DC-SIGN detects higher number of mannoses in more complex branching patterns, both can function as endocytic receptors.103,104 MMR, is expressed by microglia and astrocytes109 whereas, DC-SIGN is expressed by Dendritic cells (DC) and CNS peri-vascular cells, located on the wall of cerebral blood vessels.109 DC-SIGN binds to intra cellular adhesion molecule (ICAMs) and mannose linked Nglycans including heparan sulphate, as found on the E protein of both WNF and DV.122e127 However, DC-SIGN and MMR are unlikely candidates for the host virus entry receptors responsible for FV neuro infection, because they are neither expressed by neurons nor peripheral nerve axons.76 Furthermore, both DC-SIGN and MMR bind to DV120,126; Cytomeglavirus, Ebola and Marburg viruses all bind to DC-SIGN.103,109,122 More specifically DC-SIGN did not influence the effectiveness of neutralizing antibodies preventing the consequences of WNF infection, supporting the view that DC-SIGN is not a significant FV virus entry receptor although a polymorphism in DC-SIGN reduced DV infection.85,128 Whether or not DC-SIGN or MMR have important roles during FV infection in vivo remains to be determined.

Flaviviruses bind to a range of cellular attachment factors on host cells to increase the risk of neuroinvasion Although a specific FV virus entry receptor has not been identified, in part, due to the broad tropism of the FV in vivo and in vitro .There are several widely distributed host surface molecules (cellular attachment factors) some acting in combination that have been shown to facilitate FV attachment and entry, especially in the CNS and increase neuro invasion. Antibody binding to FV also increases their internalization and potential for infection, a process mediated by Fcreceptor on some cell types.85 The positively charged amino acids on structural protein E are attracted to negatively charged cell surface molecules such as Heparan sulphates (HSPGs) and GAGs in the cell membrane of mammalian cells.126,127 HSPGs are widely distributed, so this would not explain the limited cellular distribution of DV infection and the neurotropism of the other FVs. Heat shock proteins (HSP) 70, CD14 and a 37/ 67 kDa affinity laminin receptor, glucose regulating protein 78 (GRP78/bip) have all been proposed as putative cell attachment receptors for DV.127 Early steps in DV entry into host cells is blocked by heparin (similar to GAG) binding to amino acid clusters on the putative receptor binding

J.W. Neal domain III of E protein, similarly, chondroitin sulphate also blocked early DV entry by steric hindrance.85,126,127 This evidence supports the view that the DV E protein contains a putative receptor binding site(s) that detect widely distributed carbohydrate molecules such as GAG on host cell surface to initiate virus adsorption.101,102,128 Rather like DV, HCV cell entry involves cellular attachment factors including CD81, a tetraspanin, low density lipoprotein receptor (LDLR), Scavenger receptor class B type 1 (SR-B1) and HSPGs.129e135 Of relevance to virus entry into neurons and endothelial cells of the BBB is the data showing the HCV E protein contains Apolipo protein-E (apo-E) and this molecule interacts with cell surface LDLR to facilitate HCV attachment to host cells.130 This attachment is blocked by monoclonal antibody against apo E and treatment with heparinize prevented HCV binding to cell surface HSPGs.130 The HCV glycol proteins E1 and E2 are related to post attachment stages of HCV infection and interact with other receptors claudins, occludins, SR-B1and CD81.132e136 The FV E protein is important for initial virus attachment, this is supported by evidence that a small peptide, P9, specifically bound to WNF protein E blocked virus uptake with reduce CNS virus load.136 Heat shock protein HSP70 on neurons is also a putative receptor for JEV.110 Similarly, for JEV, changes of amino acid composition in the E protein at position 138 in domain III reduces virulence mainly by increasing virus binding to cell surface GAG and increasing clearance of JEV from the systemic circulation.99 For MEV, an amino acid change at position 390 of protein E (containing a putative FV binding domain) increased net positive charge to promote GAG dependent binding on a variety of cell types, including macrophages and this promotes clearance of systemic virus.98,99

FV neuroinfection; crossing the BBB via several routes The BBB is composed of perivascular astrocytes and micro vascular endothelial cells, whereas the CPLX and ependyma are composed of epithelial cells.137 The integrity of epithelial barriers such as the BBB relies upon the tight junctions (TJ) complexes of trans membrane proteins located on the plasma membranes of adjacent endothelial and epithelial cells. TJ contain several structural proteins, the claudins (-1, -6, -9)and occludins both associated with inter membrane proteins (zona occludens) and junctional adhesion molecules (JAD) apically located between individual epithelial and endothelial cells.138 Disruption of the TJ complexes is a well documented as an explanation for invasive properties of Influenza virus, Ebola virus, HCV and HIV; HIV-Tat down regulated occludin protein mRNA in microvascular brain cells to increase HIV neuroinvasion.139,140 The FV are able to disrupt the BBB indirectly, due to the effects of systemic inflammatory cytokines or directly by binding to structural proteins such as the claudins.138

Systemic anti-virus inflammatory cytokines also increases BBB disruption A WNF mosquito bite into skin will infect local Langerhans cells and migrate to local lymph nodes and spleen where


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7 production by BBB endothelial cells is also increased by FV infection.118,142,143 Overall, WNF infection increase systemic MMP and TNFlevels that in turndisrupt the BBB and facilitate the entry of neutrophils infected with WNF into the CNS (Trojan horse route) see Fig. 1.118,143e145 This result has been replicated, but the expression of vascular adhesion molecule and E selectin rather than MMP was responsible for trafficking of infected cells in to the CNS.144e146

they will stimulate cyto-toxic virus directed T cells, an antibody response and TNF, IL-6, IL-12 and IL-23 expression due to TLR3 and TLR7based signalling.89,115,116,121 However, the systemic anti-virus response represents a “double edged sword” because the systemic levels of TNFand IL-6 increase macrophage inhibitory factor (MIF) and adhesion molecule (ICAM) expression at the BBB and promote tight junction disruption, increasing both WNF infected leukocyte adhesion and entry into the CNS.115,116,141 In mice deficient for endothelial ICAM expression, the effects of WNF infection was less severe with both reduced BBB permeability and influx of leucocytes compared to infected wild type animals.141e144 MIF, increases nitric oxide, COX-2 prostaglandin expression and inhibition of neuronal apoptosis, further increasing virus survival. Metalo proteinases (MMP)

Neurons VV v v v v

Astrocyte Reservoir for persistent virus replication

V

V

V

Neuroinvasion of the FV in human studies identifies glycosylation status of E proteins as an important risk for

V v v Sema 7

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V v

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GAGs CS V v V HSP

v Axonal transport

vV V vv v v

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E protein glycosylation increases FV transport across the BBB with neuroinfection

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v Blood brain

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barrier

ICAM Claudins

TLR

DCSIGN

LDLR

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Figure 1 Schematic diagram showing the potential routes available to the FV(V) crossing the blood brain barrier (BBB), CPLX (choroid plexus epithelium) and glia (astrocytes and microglia). Retrograde axonal transport of virus from the skin after a FV infected mosquito bite to the CNS provides a further neuroinvasive pathway bypassing the CNS, with Sema 7 as a potential axon/ neuronal virus entry receptor. Between the individual endothelial cells are Tight Junctions (TJ) composed of claudins and other structural proteins. FV can gain accesses to these cells through virus entry receptors DC-SIGN, MMR or claudins with Low Density Lipoprotein receptors (LDLR) Scavenger B1 (SR-B1) and CD81 as co entry factors. This promotes endocytosis and microtubule trafficking of claudins to lysosomes with degradation, disrupting the TJ and allowing infected systemic cells (lymphocytes and neutrophils) into the CNS (Trojan hose). Systemic inflammatory cytokines and metalloproteinases (MMP), part of the systemic anti-viral response, also disrupt TJ with up regulation of adhesion molecules ICAM. FV infection of glia including microglia and astrocytes (strategically positioned at the BBB)can provide a reservoir for persistent infection of adjacent neurons .The FV potentially can accesses the axon at the NMJ(Neuromuscular Junction) a site of virus entry receptors including HSP (Heat shock proteins), CS (Chondritin sulphate) and GAG (Glycosaminoglycans). Sema 7a, a membrane GPI, expressed by neurons and axons has been reported as facilitating WNF entry into neurons and potentially the NMJ. Once inside the axon, retrograde axonal transport into the neuron provides an opportunity for further trans synaptic spread from neuron to neuron resulting in functionally connected populations of cells undergoing apoptosis and neurodegeneration. Please cite this article in press as: Neal JW, Flaviviruses are neurotropic, but how do they invade the CNS?, J Infect (2014), http:// dx.doi.org/10.1016/j.jinf.2014.05.010

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neuroinvasion. Flavi virus-like particle (VLP) characterized by no replication showed both neurovirulent NY99 (glycosylated E protein) and low virulent Eg101 (non glycosylated E protein) to infect endothelial cells as found in the BBB.147 Only NY99 was transported trans cellularly, without altering the integrity of the TJ similar to the findings with JEV and only after subsequent replication did the levels of TJ, JP claudins decline.148 The increased trans endothelial transport of NY99 correlates with this strain’s capacity for neuro virulence by promoting initial entry into the CNS through binding to DC-SIGN on BBB endothelial cells.149,150

FV neuroinvasion and the disruption of tight junctions (TJ) between endothelial and epithelial cells The effects of HCV, DV, WNF and JEV infection upon TJ integrity have been performed in vitro using microvascular brain endothelial cells (MVBE), epithelial cells and hepatocytes.147e154 Conflicting results in some cases are explained by the different cell types studied and methodology used to assess evidence of TJ disruption. WNF capsid protein is strategically placed in the nucleus of WNF infected glial, neurons endothelial and epithelial cells in vivo and in vitro providing an opportunity to target TJ genes.40,151,152 Infection with WNF NY99 strain did not, however, decrease claudin mRNA level, but increased specifically dynamin dependent endocytosis of claudin-1 with subsequent microtubule dependent transport to lysosomes and degradation by proteases. The reduced claudin levels disrupted the TJ and increased epithelial permeability.151,152 Pharmacological inhibition completely blocked dynamin dependent endocytosis and prevented claudin-1 degradation and blocked transport of TJ proteins to lysosomes during WNF infection.152 Similarly, for JEV in vitro infected endothelial and epithelial cells also demonstrated increased claudin-1 protein degradation and increased epithelial permeability.148 JEV replication was required to increase claudin degradation an effect independent of MMP and TNF, but dependent upon the direct effects of JEV capsid protein upon the TJ proteins.152e154

DV and HCV entry requires a multistep process and involves cellular attachment cofactors DV interacts with cellular attachment factors including claudin-1. Knock down of claudin-1 with sh RNA experiments reduced DV entry, whereas restoring claudin levels increased DV infection 1 .The interaction between the DV protein, precursor of the membrane protein (prM/M), with the extracellular loop of claudin-1resulted in the most efficient entry.155 Tight junction proteins claudins-1, -6 and -9, all facilitate HCV entry into CD81þ cells132,133 HCV entry is dependent upon E glycoprotein detection by the extra cellular loop of CD81and formation of HCV-CD81-claudin complexes,133 however, anti-CD81 antibodies did not prevent HCV endocytosis and this initial entry step relies upon other cofactors such as clatherin and dynamin for internalization.132 Neurons, astrocytes and oligodendrocytes all express claudins with CD81 present in microglia and astocytes.156e158 Therefore, virus cellular attachment

J.W. Neal factors for HCV, DV, JEV and WNF are present in neurons and glia, but also on fibroblasts, epithelium and endothelial cells.158

Evidence to support FV neuroinvasion is due to axonal transport and trans synaptic spread avoiding the BBB The selective distribution of the FV in the spinal cord and the subcortical nuclei (neurotropism) raise the possibility of FV infection spreading to the CNS via axonal transport from the periphery during viremia, as found in other neurotropic viruses such as Rabies, Polio and Herpes simplex viruses.159 This is supported by experimental data showing that BBB permeability is not increased consistently following experimental WNF infection and alternative routes for neuroinvasion must be considered.160 Peripheral inoculation of St Louis virus in Syrian hamsters resulted in viremia with infection of neurons in the olfactory epithelium with subsequent infection of the olfactory bulb and eventually the whole brain .This evidence demonstrated FV was able to enter the CNS via trans neuronal transport from the olfactory epithelium.22 Further evidence of FV trans neuronal axonal transport is provided after injecting WNFV directly into sciatic nerve, this demonstrated virus transport in anteroand retrograde directions and this resulted in anterior spinal horn neuron apoptosis and is consistent with AFP following FV neuroinfection.161,162 Colchine, a microtubule inhibitor, administered to WNF infected animals blocked axonal transport and prevented neurological signs. Extra cellular WNF virus uptake by axons was blocked by neutralizing IgG antibodies and administration of an anti E antibody 2e3 days after infection reduced paralysis.21 The importance of structural E protein providing a ligand for a putative axon receptor is supported by the failure of trans neuronal transport across synapses with a WNF reporter virus that lacked the same structural proteins.162 As yet, there is little information regarding the molecular basis for axonal transport of JEV, SLEV or MEV, although this route could explain the selective distribution of FV neuroinfection especially into the spinal cord from FV skin infection.40,50

Flavi virus neuroinfection produces primary neuronal injury An in vitro model using neuroblastoma cells and astrocytes found cells infected with WNF underwent apoptosis and cell death within 72 h independently of the presence of T cells and microglia.163,164 WNF neuron infection produces caspase 3-dependent apoptosis due to the WNF nonstructural proteins NS3 and NS8.164,165 This supports the neuropathological findings that virus transported along axons can initiate neuronal apoptosis such that neuronal death is not dependent upon a “secondary or bystander” effect due to either local activated CD8þ Tcells or FV infected microglia expressing inflammatory cytokines.166,167 WNF infects neurons through transcytosis (molecules are transported from one extra cellular space to another within endosomes), although the identity of virus entry receptors is, again, not known.19 One potential WNF neuronal virus


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entry receptor is membrane protein Semaphorin 7A (Sema 7A) this is a highly conserved membrane GPI protein, responsible for axon guidance, T cell response and regulating TGF-1 expression .In wild type Sema 7A mice, WNF inoculation produced mice neuronal infection whereas in mice deficient in Sema 7A mice inoculated with WNF were protected against WNF neuroinfection.168

Conclusions Only a small percentage of individuals infected with WNF experience neurological signs and symptoms; clinician and neuropathological data are consistent with FV having neurotropic properties involving subcortical and cortical neurons. The host immune response to FV infection is usually effective and this represents a favourable outcome between protective CD8þ T cells and neutralizing antibodies available to clear infected WNF neurons, against the potentially destructive effects of systemic inflammatory cytokines and cell adhesion factors upon the BBB .In a small number of infected individuals systemic inflammatory cytokine related disruption of the BBB provides a route for FV neuroinvasion. The identity of the host receptor(s) for FV entry into neurons, CPLX epithelium and the BBB endothelium, are not known; FV E proteins, especially when glycosylated, facilitate virus attachment to host cell membranes and this can be blocked by neutralizing antibodies against E protein epitopes .Throughout the CNS and systemic organs are distributed FV virus attachment factors and receptors (GAGs, HSPGs, CD81, claudins, DC-SIGN), that promote FV entry into neurons, glia and non-CNS tissues; they facilitate FV entry into these cells via a multi-step process, sometimes involving one or more of these attachment factors. In vitro the FV bind to and disrupt claudins located in tight junctions to promote FV transport across the disrupted BBB and CPLX, either as free virus or within infected systemic cells. Neuroinvasion is also the result of axonal transport of FV from the periphery into the spinal cord and olfactory bulb, although the identity of a host receptor or cellular attachment factors responsible for FV entry into axons and neurons in vivo is not yet known. Both examples of FV entry into the CNS, involves the targeting of widely distributed proteins related to either neuronal transport (microtubules)or BBB epithelial integrity(virus attachment factors) making the CNS especially vulnerable to FV neuro infection, once they have evaded the host’s protective anti-viral response.

Uncited reference 53,58,70.

References

1. Davies LE, DeBiasi R, Goade DE. West Nile virus neuro invasive disease. Ann Neurol 2006;60(3):286e300. 2. Tyler K. Emerging infections of the central nervous system. Part 1. Arch Neurol 2009;66:81e91. 3. Tyler K. West Nile infection in the United States. Arch Neurol 2004;61:1190e5.

9 4. Kulasekera VL, Kramer L, Nasci RS, et al. West Nile infection in mosquitoes birds and horses and humans. Staten Island New York 2000. Emerg Infect Dis 2001;7:722e5. 5. Kramer LD, Styer LM, Ebel GD. A global perspective on the epidemiology of West Nile virus. Annu Rev Entomol 2008;53: 61e81. 6. Petersen LR, Brault AC, Nasci RS. West Nile virus; review of the literature. JAMA 2013;310(3):308e15. 7. Hayes EB, Sevar JJ, Zaki SR, Lanciotti RS, Bode AV, Capbell GL. Virology , pathology and clinical manifestations of West Nile Virus disease. Emerg Infect Dis 2005;111(8): 1174e9. 8. Sevjar JJ. The long term outcomes of Human West Nile virus infection. Emerg Infect Dis 2007 Jun 15;44:1617e24. 9. Solomon T, Winter PM. Neuro virulence and host factors in flavivirus encephalitis evidence from clinical epidemiology. Arch Virol Suppl 2004;18:161e70. 10. Solomon T. Flavivirus encephalitis. N Engl J Med 2004;351: 370e8. 11. Misra KM, Kalita J. Overview: Japanese encephalitis. Prog Neurobiol 2010;91:108e20. 12. Solomon T, Kneen R, Dung NM, Khan VC, Thuy TT, et al. Poliomyelitis-like illness due to Japanese encephalitis virus. Lancet 1998;351:1094e7. 13. Solomon T, Dung TM, Keen R, et al. Seizures and raised intracranial pressure in Vietnamese patients with Japanese encephalitis. Brain 2002;1255:1084e93. 14. Gubler DJ. Dengue and Dengue haemorrhagic fever; its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G (Eds). Dengue and Dengue haemorrhagic fever. CAB 1997 International, London. p. 1e22. 15. Solomon T. Neurological manifestations of Dengue fever. Lancet 2000:25. 16. Samuel JV, Diamond M. Pathogenesis of West Nile virus infection a balance between virulence innate and adaptive immuQ3 nity and viral evasion. J Virol 2006;80(19):9349e60. 17. Monaco S, Ferrari S, Gajofatto A, Zanussso G, Maritto S. HCVrelated nervous system disorders. Clin Dev Immunol 2012: 236148. doi:1155/2012/236148. 18. Laskus T. Emerging evidence of Hepatitis C virus neuroinvasion. AIDS 2005:S140e4. 19. Suthar MS, Diamond M, Gale. West Nile virus infection and immunity. Nat Rev Microbiol 2013;11:115e27. 20. Iwamoto M, Jerigan DB, Guasch A, et al. Transmission of West Nile fever virus from an organ donor to four transplant recipients. N Engl J Med 2003;348:2196e203. 21. Monath TP, Cropp CB, Harrison AK. Mode of entry of a neurotropic arbovirus into the central nervous system; reinvestigation of an old controversy. Lab Invest 1983;4:399e410. 22. Samuel M, Hwang V, Siddharthan JD, Morrey MS, Diamond M. Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis. Proc Natl Acad Sci U S A 2007;104(43):17140e5. 23. Omalu BI, Shakir AA, Wang G, Lipkin WI, Wiley C. Fatal fulminant Pan Meningo-Polioencephalitis due to West Nile Fever. Brain Pathol 2003;13:465e72. 24. Armah HB, Wang G, Omalu B, et al. Systemic distribution of West Nile virus infection; post mortem immunohistochemical study of six cases. Brain Pathol 2007;17:354e62. 25. Gyure KA. West Nile infections. J Neuropathol Exp Neurol 2009;68(10):1053e60. 26. Zimmerman HM. The pathology of Japanese B encephalitis. Am J Pathol 1946;22:965e91. 27. Lincoln A, Siverston S. Acute phase of Japanese encephalitis two hundred and one cases in American soldiers. JAMA 1952;150:268e73. 28. Gadoth N, Weitzman S, Lehmann EE. Acute anterior myelitis complicating West Nile fever. Arch Neurol 1979;36:172e3.


63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

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10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

29. Guarner J, Shieh WJ, Hunter S. Clinicopathological study and laboratory diagnosis of 23 cases with West Nile encephalomyelitis. Hum Pathol 2004;35:983e90. 30. Jeeha LE, Sila CA, Lederman RJ, Prayson RS, Isada CM, Gordon SM. West Nile infection a new acute paralytic illness. Neurology 2003;61:55e9. 31. Li JJA, Loeb M, Shy AK, Shah AC, Tselis WJ, Kupski, et al. Asymmetric flaccid paralysis; neuromuscular presentation of West Nile Infection. Ann Neurol 2003;53:703e10. 32. Sampson BA, Ambrosi C, Harlot C, Rei ER, Veress KJ, Armbrustmachr V. The pathology of Human West Nile virus infection. Hum Pathol 2000;31:527e31. 33. Leis AA, Stokic DS, Polk JL, et al. Polio myelitis-like syndrome from west Nile virus infection. N Engl J Med 2002;347: 1279e80. 34. Leis AA, Stokic DS, Polk JL, et al. Neuromuscular manifestations of West Nile fever virus infection. Front Neurol 2012; 3:37. 35. Misra UK, Kaliata SUK, Kaliata S. Anterior horn cells are also involved in Japanese encephalitis. Arch Neurol Scan 1997; 96:114e7. 36. Savant CS, Singhal BS, Jankovic J, Khan M, Virani A. Substantia nigra lesions in viral encephalitis. Mov Dis 2003;18:213e6. 37. Suzuki M, Phillips CA. St Louis encephalitis. A histopathologic study of fatal cases from the Houston epidemic in 1964. Arch Pathol 1966;81:47e54. 38. Kienzle E, Boyes L. Murray valley encephalitis; case report and review of the radiological features. Australas Radiol 2003;47(6):61e3. 39. Knox J, Cowan RV, Dayle JS, et al. Murray Valley encephalitis; a review of clinical features diagnosis and treatment. Med J Aust 2012;196(5):322e6. 40. Douglas MS, Stephens DP, Burrow JN, Anstey NM, Talbot K, Currie BJ. Murray valley encephalitis in an adult traveller complicated by long term flaccid paralysis: case report and review of the literature. Trans R Soc Trop Med Hyg 2007; 101:284e8. 41. Varatharay A. Encephalitis in the clinical spectrum of Dengue infection. Neurol India 2010;58(4):585e91. 42. Wilkinson J, Radkowski M, Laskus T. Hepatitis C neuroinvasion: identification of infected cells. J Virol 2009;83(3): 1312e9. 43. Wilkinson J, Radkowski M, Eschbacher JM, Laskus T. Activation of brain macrophages/microglia cells in hepatitis C infection. Gut 2010;10:1394e14000. 44. Casato M, Saadoun A, Marchetti A, et al. Central nervous system involvement in hepatitis C virus cryoglobulinemai vasculitis; a multi centre case control study using magnetic resonance imaging and neuropsychological test. J Rheumatol 2005;32:484e8. 45. Kumar S, Misra UK, Kalita J, Salwani V, Gupta RK, Gujral R. MRI in Japanese encephalitis. Neuroradiology 1997;39(3):180e218. 46. Kalaita S, Misra UK, Pandey S, Dhole TN. A comparison of clinical and radiological findings in adults and children with Japanese encephalitis. Arch Neurol 2003;60:1760e4. 47. Cerna F, Mehrad B, Luby JP, Burns D, Fleckstein JL. St Louis Encephalitis and the substantia nigra: MR imaging evaluation. AMJR 1999;20:1281e3. 48. Eiseidel L, Kat E, Ravindra J, Slavaontionsck J, Gordon D. MR findings in Murray Valley encephalitis. AJNR 2003;24(7): 1379e83. 49. Haymaker W, Sabin MA. Topographical distribution of lesions in central nervous system in Japanese encephalitis. Nature of lesions with a report of a case from Okinawa. Arch Neurol Psych 1947;57:673e92. 50. Johnson RT, Burke DS, Elwel Ml, et al. Japanese encephalitis; immunocytochemical studies of viral antigen and inflammatory cells in fatal cases. Ann Neurol 1985;18(5):567e73.

J.W. Neal 51. Desai A, Shankar SK, Ravi V, Chandramuki V, Gourie-Devi M. Japanese encephalitis virus antigen in the human brain and its topographical distribution. Acta Neuropathol 1995;89: 368e437. 52. German AC, Myint KS, Mai NY, et al. A preliminary neuropathological study of Japanese encephalitis in humans and a mouse model. Trans R Soc Trop Med Hyg 2006;100(12): 1135e45. 53. Fratkin JD, Leiss AA, Stokic DS, Slavinski SA, Geis RW. Spinal cord pathology in human West Nile virus infection. Arch Q4 Path Lab 2004;128:533e7. 54. Agamanolis DP, Leslie MJ, Caveny EA, Guarner J, Shieh WJ, Zaki SR. Neuropathological findings in West Nile Virus encephalitis; a case report. Ann Neurol 2003;54:547e51. 55. Bosanko CM, Gilroy J, Wang A-M, Sanders WS, Dulai M, Wilson J, et al. West Nile Virus encephalitis involving the substantia nigra. Arch Neurol 2003;601:448e1452. 56. Van Marle G, Ostermann H, Dunham C, et al. West Nile virusinduced neuroinflammation: glial infection and caspsid protein-mediated neurovirulence. J Virol 2007;20:10933e49. 57. Thongtan T, Ceepsunthorn P, Chaiworkaul V, et al. Highly permissive infection of microglial cells by Japanese encephalitis virus: a possible role as a viral reservoir. Microbes Infect 2010;12(1):137e45. 58. Chen CJ, Ou YC, Lin SY, et al. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. J Gen Virol 2010;91(pt4):1028e103. 59. Chan-Tack KM, Forest G. West Nile meningoencephalitis and acute flaccid paralysis after infliximab treatment. J Rheumatol 2006;33:191e2. 60. Engle MJ, Diamond MS. Antibody prophylaxis and therapy against West Nile virus infection in wild type and immuno deficient mice. J Virol;77:12941e9. 61. Lanteri MC, Heitman JW, Owen B. Comprehensive analysis of West Nile fever-specific T cell responses in humans. J Infect Dis;197:1296e306. 62. Shresta B, Diamond MS. The role of CD8T cells in control of West Nile virus infection. J Virol 2004;78:8312e21. 63. Shrestha B, Samuel MS, Diamond MS. CD8þ T cells require perforin to clear West Nile fever from infected neurons. J Virol 2006;80:119e29. 64. Sitiati E, Mc Candless EE, Klein RS, Diamond MS. CD 40-CD40 ligand interactions promote trafficking of CDT 8 cells into the brain and protection against West Nile fever encephalitis. J Virol 2007;81:9801e11. 65. Shresta B, Pinto AK, Green S, Bosch I, Diamond MS. CD8þ T cells use TRAIL to restrict West Nile virus pathogenesis by controlling infection in neurons. J Virol 2012;86(17):8937e48. 66. Shresta B, Zhang B, Purtha WE, Klein RS, Diamond MS. Tumour necrosis factor alpha protects against lethal West Nile virus infection by promoting trafficking of mononuclear leucocytes into the central nervous system. J Virol 2008;82:8937e48. 67. Wang Y, Lobigs M, Lee E, Mullbacher A. CD8þ T cells mediate recovery and immunopathology in West Nile virus encephalitis. J Virol 2003;77:13323e34. 68. Zhang B, Chang YK, Lu B, Diamond MS, Klein RS. CXCR3 mediates regional-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis. J Immunol 2008;180:2641e9. 69. Garcia-Lopez MA, Sanchez-Madrid F, Rodriguez-Frade JM, et al. CXCR3 chemokine receptor distribution in normal and inflamed tissues; expression on activated lymphocytes, endothelial cells dendritic cells. Lab Invest 2001;81:409e18. 70. Christensen JE, Lemos C, Moos T, Christensen JP, Thomsen AR. CXCL10 is the key ligand for CXCR3 on CD8þ effector T cells involved in immune surveillance of the lymphocytic chorion meningitis virus infected nervous system. J Immunol 2006;176:4235e43.


63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

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71. Mc Candless E, Zhang B, Diamond MS, Klein RS. CXCL12 limits inflammation by localizing mononuclear infiltrates to the perivascular space during experimental auto immune encephalomyelitis. J Immunol 2006;177:8053e64. 72. Lim K. CCR5 deficiency is a risk factor for early clinical manifestations of West Nile fever virus infection but not for viral transmission. J Infect Dis 2010;201:178e85. 73. Lim K. Genetic deficiency in chemokine receptor CCR5 is a strong risk factor for symptomatic West Nile fever infection; meta analysis of 4 cohorts in US epidemic. J Infect Dis 2008; 197:262e5. 74. Sitati EM, Diamond MS. CD4þ T cells are required for clearance of West Nile virus from the central nervous system. J Virol 2006;80:12060e9. 75. Diamond MS, Shresta B, Marri A, Mahan D, Engle M. B cells and antibody play critical roles in the immediate defence of disseminated infection by West Nile encephalitis virus. J Virol 2003;77:2578e86. 76. Diamond MS, Sitati EM, Friend LD, Shrestha Higgs, Engle MA. Critical role for induced IgM in the protection against West Nile virus infection. J Exp Med 2003;12:1853e6. 77. Klein RS, Diamond MS. Immunological headgear ;antiviral immune response protects against neuroinvasive West Nile virus. Trends Mol Med 2008;14(7):286e94. 78. Diamond MS. Innate and adaptive immune responses determine protection against disseminated infection by West Nile encephalitis virus. Viral Immunol 2003;16:259e78. 79. Gasque P. Complement; a unique innate immuno-sensor for danger signals. Mol Immunol 2004;41:1089e98. 80. Griffiths M, Neal JW, Gasque P. The regulation of the CNS innate immune response is vital for the restoration of tissue homeostasis (repair) after acute brain injury; a brief review. Int J Inflamm 2010. 81. Gasque P, Ischenko A, Legodec J, Mauger C, Schouft M-T, Fontaine M. Expression of complement classical pathway by human glioma cells in culture. A model for complement expression by nerve cells. J Biol Chem 1993;268:25068e74. 82. Chung KM, Liszewski MK, Nybakken G, et al. West Nile non structural protein NS1 inhibits complement activation by binding to regulatory protein factor H. Proc Natl Acad Sci U S A 2006;103:19111e6. 83. Mehlhop E. Complement activation is required for induction of a protective antibody response against West Nile infection. J Virol 2005;79:7466e74. 84. Mehlhop E, Diamond MS. Protective immune responses against West Nile virus are primed by distinct complement pathways. J Exp Med 2006;203:1371e8. 85. Pierson TC, Kielian M. Flaviviruses; braking the entering. Curr Opin Virol 2013;3(1):3e12. 86. Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Premont D. Structural basis of West-Nile virus neutralization by a therapeutic monoclonal antibody. Nature 2005;437: 764e9. 87. Kaufman B, Nybaaken G, Chipman PR, et al. West Nile virus in complex with a Fab fragment of neutralizing monoclonal antibody. Proc Natl Acad Sci U S A 2006;103:12400e4. 88. Oliphant T, Engle M, Nybakken GE, et al. Development of humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med 2005;11(5):522e30. 89. Biegel JH, Nordstrom JL, Pillemer SR, et al. Safety and pharmacokinetics of a single intravenous dose of MGAWN1, a novel monoclonal antibody to West Nile virus. Antimicrob Agents Chemother 2010;54(6):2431e6. 90. Beasley DW. Vaccines and immunotherapeutics for the prevention and treatment of infections with West Nile virus. Immunotherapy 2011;3:269e85. 91. Chung KM, Nybakken G, Thompson BS, et al. Antibodies against West Nile virus non structural protein NS1 prevent

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110.

111.

lethal infection through Fc g receptor-dependent and independent mechanisms. J Virol 2006;l80:1340e51. Vogt MR, Dowd KA, Engle M, et al. Poorly neutralizing crossreactive antibodies against the fusion loop of West Nile Fever virus envelope protein protect in vivo via Fc receptor and. Shirato K, Miyosh H, iGoto A, et al. Viral envelope protein glycosylation is a molecular determinant of neuro invasiveness of the NewYork strain of West Nile Virus. J Gen Virol 2004;85(pt12):3637e45. Beasley DW, Li L, Suderman MT, Barret AD. Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 2002 Apr 25;296(1):17e23. Beasley DW, Williams MC, Hang S, et al. Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1West Nile virus strains. J Virol 2005;79:8339e47. Whiteman MC, Wicker JA, Kinney RM. Multiple amino acid changes at the first glycosylation motif in NS1 protein of west Nile virus are necessary for complete attenuation for mouse neuro invasiveness. Vaccine 2011;52:9702e10. Ni H, Barrat ADT. Molecular difference between wild-type Japanese encephalitis virus strains of high and low mouse neuro invasiveness. J Gen Virol 1996;77:1449e55. Lobbigs M, Marshall, Weir Dalgararro L. Murray valley encephalitis virus field strains from Australia and Papua New Guinea ; studies on the sequence of the major envelope protein gene and virulence in mice. Virology 1988;165(1):245e55. Lee E, Lobbigs M. Mechanisms of virulence attenuation of Glycosaminoglycan-binding variants of Japanese Encephalitis virus and Murray Valley encephalitis virus. J Virol 2002; 76(10):4901e11. Mondette JS, Lozach PY, Amara A, Gamarnik AV. Essential role of Dengue virus envelope protein N glycosylation at asparagine-67during viral protein propagation. J Virol 2007; 81(13):7136e48. Erb SM, Butrapet S, Moss KJ, et al. Domain-III FG loop of the dengue virus type 2 envelope protein is important for infection of mammalian cells and Aedes aegypti mosquitoes. Virology 2010;406(2):328e35. Vieyes G, Thomas X, Descamps V, Duverlie G, Patel AH, Dubuisson J. Characterization of the envelope glycoproteins associated with infectious hepatitis C virus. J Virol 2010;84: 10159e101682. van Kooyk T, Geijenbeek BH. DC-SIGN escape mechanism for pathogens. Nat Rev Immunol 2003;3:697e709. Cambi A, Koopman M, Figdorow CG. C-type lectins detect pathogens. Cell Microbiol 2005;7(4):481e8. Bigham AW, Buckingham KJ, Hussain S, et al. Host genetic risk factors for West Nile virus infection and disease progression. PLoS One 2011;6(9):e24745. Loeb M, Eskandrian S, Rupp M. Genetic variants and susceptibility to neurological complications following West Nile virus infection. Infect Dis 2011;204:1031e7. Glass WG, Mc Dermot DH, Lim JK, et al. CCR5 deficiency increases risk of symptomatic West Nile Fever infection. J Exp Med 2006;203:35e40. Yakub I, Lillibridge KM, Moran A. Single nuclear polymorphisms in genes for 20 -50 -oligodenylate synthetase and RNAse L in patients hospitalized with West Nile virus infection. J Infect Dis 2005;192:1741e8. Denizot M, Neal JW, Gasque P. Encephalitis due to emerging viruses: CNS innate immunity and potential therapeutic targets. J Infect 2012;65:1e16. Das S, Laxminarayana SV, Chandra N, et al. Heat shock protein 70 on Neuro 2a cells is a putative receptor for Japanese encephalitis virus. Virology 2009 Mar 1;385:47e57. Frederickersen BL, Keller BC, Forneck J, Katze MG, Gale Jr M. Establishment and maintenance of the innate antiviral


63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

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12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127. 128.

129.

130.

131.

132.

J.W. Neal response to West Nile Fever involves both RIG-1and MDA5 signaling through IPS-1. J Virol 2008;82:609e16. Nazmi A, Dutta K, Basu A. RIG-I mediates innate immune response in mouse neurons following Japanese Encephalitis Infection. PLoS One 2011;6:e21761. Suthar M, Gale M, Owen DM. Evasion and disruption of innate immune signalling by Hepatitis C and West Nile viruses. Cell Microbiol 2009;11(6):880e8. Cho H, Diamond MS. Immune response to the West Nile virus infection in the central nervous system. Viruses 2012;4: 3812e30. Wang T, Town T, Alexopoulou A, et al. Toll-like receptor 3 mediates West Nile entry into the brain causing lethal encephalitis. Nat Med 2004;10:1366e73. Daffis S, Samuel M, Suthar S. Toll-like receptor 3 has a protective role against West Nile virus infection. J Virol 2008;82: 10349e58. Suzuki T. Japanese encephalitis virus up regulates expression of macrophage migration inhibitory factor (MIF)mRNA in mouse brain. Biochem Biophys Acta 2000;1517:100e6. Aronja A, Foellmer HG, Town T, et al. Abrogation of macrophage migration inhibitory factor decrease West Nile lethality by limiting viral neuroinvasion. J Clin Invest 2007;117(10): 3059e66. Wilson JR, de Sessions PF, Leon MA, Scholle F. West Nile virus non structural protein 1 inhibits TLR3 signal transduction. J Virol 2008;82:8262e71. Szretter KJ, Daffis S, Patel J, et al. The innate immune adaptor molecule MyD88 restricts West Nile virus replication and spread in neurons in the central nervous system. J Virol 2010;23:12125e38. Town T, Bai F, Wang T, et al. Toll-like receptor 7 mitigates lethal West Nile encephalitis via interleukin23-dependent immune cell infiltration and homing. Immunity 2009;20: 242e325. Alvarez C, Lasala F, Carrillo J, et al. Type lectins DC In-SIGN and L-SIGN mediate cellular entry Ebola virus in cis and trans. J Virol 2002;76(13):6841e4. Martina B, Koraka P, van den Doel P, et al. DC-SIGN enhances infection of cells with glycosylated West Nile virus in vitro and virus replication in human dendritic cells induces production of IFN-alpha and TNF-alpha. Virus Res 2008;135:64e71. Taasaneetritthep B, Burgess TH, Granelli-Pipero A, et al. DCSIGN(CD209) mediates Dengue virus infection of human dendritic cells. J Exp Med 2003;197(7):823e9. Miller JL, de Wet BJ, Martinez-Pomares L, et al. The mannose receptor mediates dengue virus infection of macrophages. PLoS Path 2008;4(2):e17. Chen Y, Maguire T, Hileman RE, Fromm JR, Esko JD, et al. Dengue virus infectivity depends upon envelope protein binding to target heparan sulfate. Nat Med 1997;3(8):866e71. Kazuya IP, Hidari J, Abe T, Suzuki T. Carbohydrate-related inhibitors of Dengue virus entry. Viruses 2013;5(2):605e18. Obara CJ, Dowd KA, Ledgerwood JE, Pierson TC. Impact of viral attachment factor expression on antibody-mediated neutralization of flaviviruses. Virology 2013;1(437):20e7. Fletcher NE, Wilson GK, Murray J, et al. Hepatitis C virus infects the endothelial cells in the blood brain barrier. Gastroenterology 2004;142:634e43. Jiang J, Cun W, Wu X, Shi Q, Tang H, Luo G. Hepatitis C Virus attachment mediated by Apolipoprotein E binding to cell surface heparin sulphate. J Virol 2012;86:7256e67. Chen WR, Tesh RB, Rico-Hesse R. Genetic variation of Japanese encephalitis virus in nature. J Gen Virol 1990;71: 2915e92. Harris HJ, Davis C, Mullins JG, et al. Claudin association with CD81 defines Hepatitis C virus entry. J Biol Chem 2010;27: 21092e102.

133. Farquhar M, Hu K, Harris HJ, et al. Hepatitis C virus induces CD81 and claudin-1 endocytosis. J Virol 2012;8:4305e16. 134. Morikawa K. The roles of Cd81 and glycoaminoglycans in the adsorption and uptake of infectious HCV particles. J Med Virol;79:714e23. 135. Scarselli B. The human scavenger receptor class B type 1 is a novel candidate receptor of Hepatitis C virus. EMBO J 2002; 21:5017e25. 136. Bai F, Town T, Pradhan D, et al. Antiviral peptides targeting the West Nile virus envelope protein. J Virol 2007;81(4): 2047e55. 137. Pachter JS, de Vries HE, Fabry Z. The blood-brain barrier and its role in immune privilege in the central nervous system. J Neuropathol Exp Neurol 2003;62:593e604. 138. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Chapter 12. Intracellular compartments and protein sorting. Molecular biology of the cell. 4th ed. Garland Science; 2002. pp. 659e710. 139. Meeretens L, Bertaux C, Cukierman L, et al. The tight junction proteins Caludin-1, -6 and -9 are entry cofactors for Hepatitis C virus. J Virol 2008;82:3555e60. 140. Xu R, Feng X, Xie X, Zhang J, Wu D, et al. HIV-1 Tat protein increases the permeability of he brain endothelial cells by both inhibiting occludin expression and cleaving occludin via matrix metalloproteinases. Brain Res 2012;1436:13e9. 141. Dai J, Wang P, Bai Town T, Fikrig E. ICAM-1 participates in the entry of West Nile virus into the central nervous system. J Virol 2008;82:4164e416. 142. Gidday JM. Leukocyte-derived matrix metallo protein ases-9 mediated blood brain barrier breakdown and is proinflammatory after transient focal cerebral ischemia. Am J Heart Physiol Circ Physiol;289:H558e68. 143. Wang P, Dai J, Bai F, et al. Metalloproteinase 9 facilitates West Nile virus entry into the brain. J Virol 2008;82:8978e85. 144. Bai F. A paradoxical role for neutrophils in the pathogenesis of West Nile virus. J Infect Dis 2010;202:1804e12. 145. Roe K, Kumar M, Lum S, Orillo B, Nerukar VR, Verma S. West Nile virus-induced disruption of the blood brain barrier in mice is characterized by the degradation of the junctional complex proteins and increase in multiple metalloproteinases. J Gen Virol 2012;93:1193e11203. 146. Shen J, T-To SS, Schreiber L, King NJ. Early e-selectin V-CAM1, ICAM-1, and late major histocompatibility complex antigen induction in human endothelial cells by flavivirus and co modulation of adhesion molecules expression by immune cytokines. J Virol 1997;71:9323e32. 147. Hasebe R, Suzuki T, Makino Y, et al. Trans cellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein. BMC Microbiol 2010;8(10):2180e91. 148. Agrawal T, Sharavani V, Nair D, Medigeshi G. Japanese encephalitis virus disrupts cell cell junctions and affects epithelial permeability barrier functions. PLoS One 2013;8(7): e699465. 149. Davies CW, Mattei LM, Nguyen HY, Anasarah-Sobrinho RW, Doms RW, Pierson TC. The location of asparaginase-linked glycans on West Nile virions controls the interaction with CD209 Dendritic cell-specific ICAM-3 grabbing non integrin. J Biol Chem 2006;282:37183e43719. 150. Dropulic B, Masters CL. Entry of neurotropic arboviruses into the central nervous system: an in vitro study using mouse brain endothelium. J Infect Dis 1990;161(4):885e89141. 151. Medigeshi GR, Hirsch AJ, Brien JD, et al. West Nile Virus capsid degradation of claudin protein disrupts epithelial barrier function. J Virol 2009;83:6125e34. 152. Xu Z, Waeckerlin R, Urbanowski MD, van Marle G, Hobman TC. West Nile Virus infection causes endocytosis of a specific subset of tight junction membranes. PLoS One 2012;7(5):e37886.


63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124

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Flaviviruses are neurotropic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

153. Verma S, Kumar M, Gurajav U, Lum S, Nerukar VR. Reversal of West Nile virus induced blood brain barrier disruption and tight junction protein degradation by matrix metallo proteinases inhibitors. Virology 2010;397:130e8. 154. Verma S, Lo Y, Chapagain M, et al. West Nile virus infection modulates human brain micro vascular endothelial cells tight junction proteins and cell adhesion molecules; transmigration across the in vitro blood brain barrier. Virology 2009;15:385. 155. Chen P, Tang H, Li Q. The interaction between claudin-1 and dengue viral protein prM/Mprotein for its entry. Virology 2013;446(1e2):303e13. 156. Romanitan MO, Popescu BO, Spubler S, et al. Altered expression of claudin family proteins in Alzheimer’s disease and vascular dementia. J Cell Mol Med 2010;14(5):1088e100. 157. Djikstra S, Geisert EE, Dijkstra CD, Bar PR, Joosten EA. CD81 and microglial activation in vitro: proliferation phagocytosis and nitric oxide production. J Neuroimmunol 2001;114: 151e9. 158. Cao J, Zhao P, Zhao LJ, Wu SM, Zhu SY, Qi ZT. Identification and expression of human CD81 gene in murine NIH/3T3cell membrane. J Microbiol Methods 2003;54(1):81e5. 159. Salinas S, Schiavo G, Kremer EJ. A hitch hikers guide to the nervous system; the complex journey of viruses and toxins. Nat Rev Microbiol 2010;8:645e55. 160. Morey JD, Olsen AL, Siddharthan V, et al. Increased blood brain barrier permeability is not a primary determinate for lethality of West Nile virus infection in rodents. J Gen Virol 2008;89(pt2):467e73.

13 161. Morrey JD, Siddhartan V, Wang H, et al. West Nile virusinduced acute flaccid paralysis is prevented by monoclonal antibody treatment when administered after injection of spinal cord neurons. J Neurovirol 2008;14(2):152e70. 162. Wang H, Siddharthan V, Hall JO, Morrey JD. West Nile virus preferentially transports along motor neuron axons after sciatic nerve injection in hamsters. J Neurovirol 2009;15(4): 293e6. 163. Shresta B, Gottlieb D, Diamond MS. Infection and injury of neurons by West Nile Encephalitis virus. J Virol 2003;77(24): 13203e13. 164. Samuel MA, Morey JD, Diamond MS. Caspase 3-dependent cell death of neurons contributes to pathogenesis of West Nile Virus encephalitis. J Virol 2007;81:2614e23. 165. Ramanatan MP, Chambers JA, Pankhong P, et al. Host killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase 8-based apoptotic pathway. Virology 2006;345:56e72. 166. Chen CJ, Chen JH, Chen SY. Up regulation of RANTES gene expression in neuroglia by Japanese encephalitis virus infection. J Virol 2004;78(22):12107e9. 167. Goshal A, Das S, Ghosh S, Mishra MK, et al. Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis. Glia 2007;55(5):483e96. 168. Sultana H, Neelakanata G, Foellmer HG, et al. Semaphorin 7a Q1 contributes to West Nile virus pathogenesis through TGF Q6 b1/Smad 6 signalling. J Immunol 2012;189:3150e8.


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