Biomarkers in Japanese Encephalitis: A Review

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 591290 24 pageshttpdxdoiorg1011552013591290

Review ArticleBiomarkers in Japanese Encephalitis A Review

Ravi Kant Upadhyay

Department of Zoology D D U Gorakhpur University Gorakhpur Uttar Pradesh 273009 India

Correspondence should be addressed to Ravi Kant Upadhyay rkupadhyayahoocom

Received 24 August 2013 Revised 16 October 2013 Accepted 21 October 2013

Academic Editor Antoni Camins

Copyright copy 2013 Ravi Kant UpadhyayThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

JE is a flavivirus generated dreadful CNS disease which causes high mortality in various pediatric groups JE disease is currentlydiagnosed by measuring the level of viral antigens and virus neutralization IgM antibodies in blood serum and CSF by ELISAHowever it is not possible to measure various disease-identifying molecules structural and molecular changes occurred intissues and cells by using such routine methods However few important biomarkers such as cerebrospinal fluid plasma neuro-imaging brain mapping immunotyping expression of nonstructural viral proteins systematic mRNA profiling DNA and proteinmicroarrays active caspase-3 activity reactive oxygen species and reactive nitrogen species levels of stress-associated signalingmolecules and proinflammatory cytokines could be used to confirm the disease at an earlier stage These biomarkers may alsohelp to diagnose mutant based environment specific alterations in JEV genotypes causing high pathogenesis and have immensefuture applications in diagnostics There is an utmost need for the development of new more authentic appropriate and reliablephysiological immunological biochemical biophysical molecular and therapeutic biomarkers to confirm the disease well in timeto start the clinical aid to the patients Hence the present review aims to discuss new emerging biomarkers that could facilitatemore authentic and fast diagnosis of JE disease and its related disorders in the future

1 Background

Japanese encephalitis virus is a single stranded positive senseRNA virus belonging to family Flaviviridae It is one ofthe major causative agents of pediatric encephalitis or viralencephalitis in Southeast Asia Due to demographic envi-ronmental and therapeutic reasons its outbreak commonlyoccurs almost every year among children [1] JE is a dreadfulzoonotic disease that generates high morbidity and mortalityin pediatric groups Its transmission is seasonal that occursvery fast in rainy season due to mass breeding of rice fieldmosquito vector that isCulex tritaeniorhynchus summorosusespecially in undeveloped rural areas Because of scatteredoccurrence of JE in different states and regions in India theactual JE burden and magnitude of infection cannot easilybe estimated Due to lack of proper and timely diagnosisof JEV and extra delay in treatment very high mortalityoccurs in various infant groups [2] However JE control couldbe possible only by strengthening diagnostic facilities forits confirmation in hospitals situated in rural areas and byestablishing national surveillance system for knowing post-vaccination adverse effects Moreover an earlier diagnosis

of the disease and medical care is required for patientsNevertheless patients with mixed and typical symptoms ofbrain fever or saddleback fever with low platelet counts andhematocrit values should provide immediate clinical care[3] This clinical phase is highly important to diagnose thedisease To date a definitive diagnosis of JE can only be madewith clinical symptoms biochemical profiles and serologicalexamination of JE patients Therefore treatment strategiesmight be most effective before virus pathogenesis spreadsthroughout brain and spinal cord Thus an earlier diagnosisbased on reliable biomarkers is essential to identify the statusand intensity of JE infection

The current literature and clinical investigations revealthat climate induced genotypic variations are going on innewly emerging molecular variants of flaviviruses mainlyrelated to encephalitis It proves that JE virus is mak-ing possible modifications in antigenic structure throughmutations both in nucleotide and protein sequences It isvery hard to explain environment-induced mutations andother molecular changes occurring in antigenic sites Ifidentifying these minor differences in mutant strains of JEvirus could help to detect environment-specific alterations

2 BioMed Research International

in pathogenesis in recent past and future Further vaccineattenuated sequence alterations are significantly increasingwhich are not only changing the etiological features of thevirus but are also inducing high neurovirulence and hostimmune responses and affecting disease transmission in bothendemic and nonendemic population However other thanJE virus endemic strains new vaccine-derived recombinantstrains of JE virus have been evolved which are detected inserum samples of patients from endemic regions Howeveroverall changes occurred in JEV have increased the infectionpathogenesis and mortality rate It has also led to an increasein expansion of severity of infection in nonendemic areasAmong other possible reasons of expanding its area areglobal climates ecological and socioeconomic changes andmutations acquired by local strains of JE virus

Though so many methods of JEV detection are availableto days it is very hard to confirm the JE disease based onseeing visible pathological symptoms in patients However itis very clear that disease history of JE infection virus expo-sure and certain clinical features cannot help to diagnose thisdisease properlyThere is an utmost need to develop potentialbiomarkers which might essentially improve the diagnosisand accelerate the development of new vaccinesMore specif-ically biochemical molecular and immunological tools areconsidered to be more authentic and reliable to detect the JEvirus in different tissues and cells [4] These markers couldfacilitate fast diagnosis of JEV invasion in neuronal cellsand behavioral impairments in patients and help to confirmprogression of disease However a clinical diagnosis based ondetermination of the level of virus IgM antibodies expressionof nonstructural viral proteins and pathological changes inneuronal progenitor cells brain cortex thalamus hippocam-pus and striatum will be more helpful in JE confirmation[5] Similarly measurement of the level of certain markers inCSF such as the level of interleukins complements cytokinesand intracellular virus proteins was also found to be usefulin predicting the JEV generated risks and prevalence ofpathogenesis [6] However definitive diagnosis of JE basedon clinical symptoms serology and biochemical profile ofthe virus is quite investigative [7] but it is very difficult todiagnose on going mutations in newly emerging genotypesof JE virus circulating in the endemic population Furthergenotype-based neurovirulence antigenicity pathogenesisand mortality must reinvestigated by using new candidatemarkers Biomarkers will assist in exploration of pathologicalmechanisms followed by virus during invasion persistenceand clearance Therefore strong molecular and proteinbiomarkers are needed for the confirmation of genotype-based infection of JE virus in patients as well as its therapeuticessentiality accordingly

However biomarkers based on measurement of a highlyspecific biomolecule that may confirm the presence of JEvirus pathogen inside host will clearly display the physiolog-ical state of patient Biomolecules synthesized in response toJE virus attack and cellular invasion will show clear biologicalcharacteristics whose measurement in body fluid cells andtissues will confirm the viral disease [4 5] For exampleformation of C-reactive protein (CRP) in response to JEvirus attack works as a marker for inflammation Further

all affected specific cells molecules or genes gene productsenzymes or hormones complex organ functions charac-teristic changes that occurred in the biological structureand function of Biomolecules could serve as biomarkersTherefore biomarkers that clearly indicate and reflect theseverity and status of viral infection in patents will bemore appropriate More specifically such biomarkers couldcorrelate virus-induced changeswith the risk and progressionof JE disease and susceptibility of virus to a given treatmentSimilarly imaging biomarkers (CT PET and MRI) andnonimaging biomarkers could also explore biophysical prop-erties of virus and bio-molecules with their measurements inbiological samples (eg plasma serum cerebrospinal fluidbrain biopsy)

More specifically nucleic acid-based biomarkers suchas gene mutations or polymorphisms and quantitative geneexpression analysis virus-induced peptides proteins lipidsmetabolites and other small molecules could help in earlydiagnosis of JE However gene-based biomarker will be moreeffective and an acceptable marker and may prove better JEdiagnostic tools for fast clinical assessment of JEV in thefuture Furthermore subset of markers that might discoversecretes of virus invasion and cellular pathogenesis by usinggenomics proteomics technologies or imaging technolo-gies would play major roles in medicinal biology Furtherstrong therapeutic pharmacodynamic (PD) biomarkers willbe required for decision making and drug developmentpharmacological response and dose optimization Howeverto have an early clinical investigation ready highly specificbiomarkers are essentially required to display the JE infectionand physiological state of patient before starting certainmedicare Hence in the present review article newly emerg-ing biomarkers for JE are highlighted with their diagnosticefficacy specifications authentication and working accuracywith an objective to recognize cellular abnormalities occur-ring in the JE infected patientsThese biomarkersmight proveto be significant diagnostic tools for JE confirmation in thefuture

2 Cerebrospinal Fluid Biomarkers

JE virus cultured in the host body cells releases somany com-ponents in the cerebrospinal fluid which serve as biomark-ers Hence both interacting and noninteracting moleculesreleased by the cells in anticipation to virus load and invasionof blood and nerve cells are determined accurately Howeveralot of virus eradicating molecules are released by immunecells and virus toxinsantigens in the CSF are assayed forevaluation of presence of virus intensity and rate of infectionin humanhosts However for detection of JE virus interactingmolecules in cerebrospinal fluid and blood serum samplesare collected from both JE endemic and epidemic areasIn normal population blood serum samples are collectedin the premonsoon season and at the time of outbreakDuring infection season samples are collected twice at atime interval of ten days Patient information should berecorded with 5ndash10 different specifications it should includelocation name age sex vaccinated or not date of onset of

BioMed Research International 3

symptoms types of specimen date of collection economicgroup viability and medicare given Experienced personnelunder aseptic conditions should do sample collection CSFcollection should be done in separate vials for each bioassayrelated to biochemistrymicrobiology and virology of the JEVNormally for each investigation 05ndash1mL of CSF is requiredThe sample must be kept at 4∘C for short-term storage butfor long-term storage this sample must be kept below minus20∘CRepetitive freezing and thawing should be avoided Liquidnitrogen containers are used for transportation of samples tothe investigating laboratories

However level of virus neutralization antibodies in CSFworks as one of the important biomarkers for knowing thepresence of JE virus or any other viral infection For mea-surement of neutralizing antibodies level in cerebrospinalfluid (CSF) and serum few conventional techniques suchas viral neutralization hemagglutination inhibition (HI)complement fixation tests and immune-florescent stainingmethods are mostly applied All these techniques showedlimitations as routine diagnostic tests because these arelabor-intensive expensive cumbersome and not sensitiveto the detected antibodies in CSF [5] These virus neu-tralization antibodies successfully block viral infection byneutralization after formation of virus- antibody complexThis complex can prevent viral infection in many ways Butsuccess rate in clinical specimens remains less because of thelow level of viremia and rapid development of neutralizingantibodies (Table 1) [8] Recently much potent neutralizinghuman antibodies have been synthesized against JE virusto measure the level of circulating virus in CSF and otherbody fluids of patients These neutralizing human antibodieshelp in more accurate sero-diagnosis of JE virus in clinicalsamples [5] In addition to this rapid micro-neutralizationtest (MNT) is also developed to detect neutralizing antibodiesto JEV virus in the CSF of the Japanese encephalitis patients[4] It also helps to establish single virus infection in patients[9] It is performed by using maximum dilution of antibodywhich can confirm 90 reduction in viral infectivity aftervirus neutralization [10] Similarly reduction neutralizationtest (PRNT) helps to detect humoral immune responsegenerated after immunizationwith JE inactivated vaccine [6]Thus both MNT and PRNT detect Japanese B encephalitisvirus (JEV) neutralizing antibody titers more efficientlyand these titers work as strong markers of JE disease [11]These tests also confirm low JE infection level by measuringneutralization antibody titer [12] However early and highneutralizing antibody responses are crucial for preventingviral neuroinvasion and host fatality [13] At this stage virusgenerated Biomolecules if assayed may work as strong anduseful marker to diagnose the JE disease at an earlier stageHowever virus neutralization antibodies mainly polyclonalantibodies can subvert the attack of JE virus [7]

Further the presence of viral encephalitis is also detectedby analyzing titers of neutralizing antibodies by DEIA andother techniques such as CT MRI EEG spinal tap andbrain biopsy (Table 1) Further neutralizing antibodies syn-thesized against nonstructural neurovirulent proteinsmay bemore helpful in disease diagnosis These antibodies remaindetectable in CSF and blood within 7 days after onset

of disease [14] by IgM capturing ELISA more accurately[15] and authentically (Table 1) [16 17] Few other meth-ods like immunological haemagglutination inhibition [18]and complement fixation tests accurately detect presence ofJE infection Further immunotyping techniques based onantibody absorption precisely determine minor variationsin various immunotypes prepared against different virusantigens [19] For example by using this method 2 strains ofNakayama-NIH and Nakayama-Yakken immunotypes wereidentifiedTheNakayama-RFVL strain was found to have thecharacteristics of both immunotypes while I-58 immunotypediffers more markedly from related arboviruses such as theMurray Valley encephalitis virus and the West Nile Eg101strain Moreover the HI test is widely used for the diagnosisof Japanese encephalitis virus but shows great limitation andfail to detect cross-reactivity with other flaviviruses Morespecifically sera treated with acetone or kaolin and thenadsorbed with homotypic RBCs are used to remove anynonspecific haemagglutinins [18] (Table 1)

However glycoprotein E (V3) of different viruses whichexpresses antigenic determinants and its differential bindingto antibodies is used in haemagglutination (HA) assayFurther display of antigenic relationships of the E proteinamong several flaviviruses that is WN subgroup viruses(JE MVE WN and SLE) and other subgroup flaviviruses(DEN) MAbs prepared against the E protein of JE virus isused [19] More specifically epitopes of E protein of severalflaviviruses such as JE virus [20] tick-borne encephalitis(TBE) virus [21 22] SLE virus [23 24] YF virus [25]WN virus [26] and DEN virus [27] are used to synthesizedifferent virus-specific Mabs These antibodies can explorecross-reactive sites in genotypes and authenticate presenceof JEV among all the flaviviruses more strongly These willalso help in clinical establishment of the cause of JE diseasein particular areas Moreover serial measurement of serumNS and E proteins might be a useful marker for diagnosingthe disease and therapy A plasmid DNA vaccine encodingprM-E protein from the JE virus also elicits cellular immuneresponses Further significant homology in amino-terminalamino acid sequences of E proteins of different flaviviruses byusing amino acid sequencing and proteome analysis can alsoestablish status of antigenicity neurovirulence and diseasepathogenesis [28]

However ELISA is a more accurate method which candetect nanogramquantity of JE specific antibodies in patientrsquosblood serum and replaces both serum neutralization andHI tests because of its high sensitivity and binding to virusantigens [8] Moreover fast diagnostic automation JE ELISAtest is also developed and authenticated to use antigen JERAJERA is a recombinant antigen that consists of a stretchof peptides from different parts of JEV antigens [29] It isused as a rapid serological marker for detection of JEV-specific antibodies in patientrsquos blood Similarly another JEVIgG ELISA is also developed It is a two-step sandwich-type immunoassay (Table 1) relativelymore rapid and reliablemethod that can accurately diagnoses JE virus with a singlespecimen collected during acute phase [30] It is used in fieldevaluation of circulating JE virus among encephalitis patients[31] and is routinely used as serological in-house assay to


Table 1 Different JEV specific tests for clinical diagnosis of Japanese encephalitis virus in body fluid cells and tissues of patients

Method Sensitivity Confirmatory diagnosis

IgM capture ELISA (enzyme linkedimmunosorbent assay)DEIA dipstick enzyme linkedimmunosorbent assayPanbio JE-DEN IgM Combo ELISAJE-Chex IgM capture ELISA

Highly sensitive and confirmatorySample 5 120583L total time 135 minutes antigencoated plateHRP and tetramethyl benzidine (substrate)Highly sensitive and confirmatoryDetection of JEV positive and negativeresults in CSF and serum

Can differentiate infection type intensity andpresence of JEV strains and detects specificIgM in the cerebrospinal fluid or in the bloodDetection of immunoglobulin in human serumto JEV- derived antigens In vitro diagnostic useStatus of viral encephalitis neuroinflammation

MAC-ELISA MAC ELISA is used to diagnose secondaryflavivirus infection

Used to detect true positive and true negativesensitivity and specificity in JEV affectedpatients

Single TaqMan assay Highly sensitive and confirmatory Diagnoses virus antigensImmunofluorescent test Highly sensitive and confirmatory Works as a valuable alternative to the

established methods in detecting anti-JEVantibodies after vaccination in travelers andhelps in the diagnosis of acutely infectedpersons in vitro labeling of NPC cells

Fluoresecent markers are usedFluorescent dye 7-ADD binds to DNAThe labelled cells are detected by FL-1channel by FACS and are analyzed byusing Cell Quest Pro software to quantifypercentage of labelled cells

The amount of flouresecent antibody boundto each cell can be quantified

Florescence resonance energy transfer(FRET) Highly sensitive Detects interaction of antigens in cells

Plaque reduction or JEV-antibodyneutralization test (PRNT)

Moderately sensitive and confirmatoryOn incubation the antibody forming cellsrelease immunoglobulin which coats thesurrounding erythrocytes Complementcauses lysis of coated cells and plaque clearor red cells are counted Hemolyticefficiency of IgM antibodies is detected

Can detect humeral immune responsegenerated after immunization with JEinactivated vaccineUsed for viremia determinationPercentage neutralization is calculated fromnumber of plaques obtained

Microcomplement fixation test Moderately sensitive Can detect cellular factors and antigensVirus overlay protein binding assay(VOPBA) Highly sensitive and confirmatory Detects JE virus receptor molecules on the cells

YUNEL assay Highly sensitive Apoptosis cell membrane disruption andmorphology

Lumbar puncture test and CSF analysis Moderately sensitive and confirmatory Probable and confirmed JE

MRI (magnetic resonance imaging) Moderately sensitive and confirmatory

Can locate bilateral thalamic lesions withhemorrhage and any abnormality generated inbasal ganglia putamen pons spinal cord andcerebellum may also show pathologicalabnormalities

CT scan (computed tomography) Highly sensitive and confirmatory Can locate hyperintense lesions in the areas ofthe thalamus cerebrum and cerebellum

EEG (electroencephalogram) Moderately sensitive and confirmatory Reveals diffuse and burst suppression

CBC (complete blood count)Confirms the presence of JEV infection inchildren and helps in clinical analysis ofblood parameters

Detect leukocytosis leucopenia anemia andthrombocytopenia and supportivelymphocytic pleocytosis

Platelet count Sensitive and supportive for clinical analysis Can detect effect of fever on blood platlets

Hemagglutination inhibition test (HA)Moderately sensitiveAgglutination is done by using antigencoated particles

Antibody detection to detect rheumatoidfactors identification of antibodies to solubleantigens HA is used to detect JEV in variouspassages

Compliment fixation test (CF) orcrosslinking of antigens Moderately sensitive

Antibody detection Surface antigens aredetected by using labeled antibodies Bothmonovalent and divalent antibodies are used

Immunotyping Highly sensitive and confirmatory Differentiates genotypes of JE virus


Table 1 Continued

Method Sensitivity Confirmatory diagnosisRPHA IFA immunoperoxidase Moderately sensitive Antigen detection

Immunoblotting Highly sensitive

JEV generated infection in NCPs andrecognizes decrease in the number of colonyforming neurosphere and their self-revealHRP PBS-T

IDD (immunodouble diffusion test) Moderately sensitive

Immunologic relationship between theantigens related or indicative or unrelatedPrecipitate forms an opaque line in thecross-reactive region

Cell death assay (annexin-propidiumiodide staining test) Highly sensitive

Can recognize apoptotic cell death in controland JEV infected cells FITC labeled annexinand propidium iodide are used

Nephrometry Moderately sensitive

Antigen and antibody dilutions are used tocreate cloudiness and greater sensitivity can begenerated by using monochromatic light froma laser and by adding PEG to solution toincrease the size of aggregation

Neutralization tests Moderately sensitiveNeutralization antibody titre in sera and in CSFcan recognize homologous virus the challengevirus and the selected wild-type JE virus

Flow cytometry (FACS) Highly sensitive Intracellular signaling of JEV antigen to detectpercentage of anti-JEV-FITC positive cells

Immunohistochemistry Highly sensitive Intracellular localization of NS3 by using anti-JEV antibodies

Precipitin test Moderately sensitive Quantitative analysis of antigen and antibodyinteraction

SRID Highly sensitive To know the amount of antigen in unknownsamples

Neurovirulence test Highly sensitiveTo detect histopathological recognition of JEVpathogenesis in brain and in associated tissuesPrediction of level and cause of neurovirulence

Anticomplementarity Test Highly sensitive Identification of lesion scoresHemolysin test Moderately sensitive Percent of hemolysis in RBCsDNA microarray Highly sensitive and confirmatory Expression of genes and proteins

Site directed mutagenesis Highly confirmatory

Detects amino acid substitutions in E NS1 andNS2 proteins clone-specific substitutions andheterogeneity substitutions and is used todetect possible mutations in structural and nonstructural viral proteins

Real-time polymerase chain reaction(RT-PCR) Highly sensitive

Target sequences can be detected in genes andviral genome Amplification of immunotypestrain cloning and expression of NS3 gene ofNS3 protein of JEV

RNA studies oligonucleotideprimer-based detection of JEV functionalsequences in different genes and genecopies

Highly sensitiveDetects molecular pathogenesis at the level ofenzymes genes factors and proteins Synthesisand secretion of JEV-induced proteins

lowastPresence of JEV viral and virus secreted antibodies are detected in cerebrospinal fluid (CSF) and serum samples For component-based detection of JEV a widevariety of conventional techniques such as viral neutralization hemagglutination (HI) and complement fixation and immunoflourescent staining are usedLaboratory diagnosis of JE virus is mostly confirmed by immunological molecular and biophysical methods Most of the laboratory-based tests and clinicaldiagnostic tests are routinely used to detect presence of JEV virus and its pathogenesis but all such tests are labor-intensive expensive and cumbersome


measure JEV specific IgM antibodies However flavivirusgroup shows intense cross-reactivity to IgG level but secretedIgM level in CSF can detect JE virus more accurately [30]

Similarly a dipstick enzyme-linked immunosorbantassay is also used for detection of JE virus-specific IgMantibodies [8] (Table 1) It shows very high sensitivity andspecificity to JEV and is used as a promising diagnostic tool infield conditions It is routinely used for laboratory diagnosisof JE virus It is a simple rapid test and requires no specializedequipment Similar to dipstick ELISA MAC-ELISA is alsoused as a valuable diagnostic tool that detects secondaryflavivirus infection in comparison to hemagglutination inhi-bition test [8] It was found to be very sensitive and highlyspecific with more than 90 confidence MAC assays haveone great advantage over conventional indirect assays basedon IgG antibodies IgM detection shows higher sensitivityin MAC ELISA which shows ratio of 1 300 in diseasedand in apparent infection [32] MAC ELISA clearly providesdifference among JEV and DEN virus IgM antibodies anddiagnosis can be made from a single sample (preferably CSF)collected during early acute phase of infection [30] SimilarlyNIMHANS Bangalore NII New Delhi and K G MedicalCollege Lucknow developed JEV diagnosing kit JEV-Chexunder DBT umbrella program Chex is a rapid ELISA kitfor the detection of IgM antibodies in human CSF andserum Similarly few commercial JE detection kits such asEuroimmun anti-JEV IgM IIFT and the Panbio JapaneseEncephalitismdashDengue IgM Combo ELISA are also availablein the market which can detect JE virus infection moreaccurately These bioassays show more than 90 specificityand sensitivity These are highly reliable established methodsto detect JEV infection in travelers and common people(Table 1) However IgM antibody level is one of the importantmarkers which can more precisely investigate JE virus inacute phase of infection in CSF of patients [30] More usuallymeasurement of the rates of infection can be determined byobserving seropositivity in mosquitoes and birds

3 Serum Biomarkers

Serum protein profiles work as potential biomarkers forknowing infectious disease status in animals [33] These aregenerated by SELDI-TOF mass spectrometry in combina-tion with the ProteoMiner technology that accurately dis-plays low-abundance proteins responsible for virus infectionThese also clearly display status infectious disease and rateof infection in separate models or hosts Similarly potentialbiomarkers for a number of human and animal diseasesare facilitated by proteomic analysis of serum proteins andenzymes [10] However comparative proteomic analysis ofserum proteins on SELDI-TOF-MS [11] and chip arrayscould find differences in virus and host secreted proteinsduring various interaction periods Such arrays could bindintact proteins present in biological samples such as bodyfluids or tissue extracts and detect virus-induced effectsSuch arrays may vary in their surface chemistry for instancethey may have hydrophobic or hydrophilic properties andthereby selectively bind proteins that could be identified

by their specific molecular weights However correlationbetween SELDI-TOF MS results and clinical data couldrecognize significant variation in virus specific proteinscomponents that differ in abundance between groups ofsamples Therefore quantitative data of high- and low-abundant serum protein components measured by SELDI-TOF-MS can be used for early detection and diagnosis of viralinfectious diseases These protein profiles could alternativelyobtained in other biological samples like saliva urine orfeces Further recent developments that occurred in thefield of micro- and nanotechnology created larger interestfor researchers to develop sophisticated electronic devicesfor clinical health monitoring However several promisingprototypes are emerging in human biomedicines mainly fordiagnosis of patients with neurological diseases [23]

A quantitativemicrocomplement fixation test also detectsnanogram quantities of antigen in serum blood samples of JEvirus infected patient The test is used to detect the presenceJE virus specific antibodies in serum and is highly repro-ducible Normally after seven days of transmission of JEVvirus B cells produce antibodies during an active infectionwhich defend the body against viruses and other foreignsubstances called antigens If the antibodies are present theyattach to the antigen This combination activates or ldquofixesrdquocomplement The test is more useful to know the rate ofinfection by determining the level of serum antibodies withthe help of specific antigen Complement binds to antigen-antibody complex and leads to cell lysis Complement subse-quently binds to this antigen-antibody complex formed andwill cause the red blood cells to lyse [34] If the patientrsquos serumdoes contain a complement-fixing antibody a positive resultwill be indicated by the lack of red blood cell lysis Besidesthis measurement of glucose protein level and mononuclearwhite blood cell counts done in CSF samples which wereobtained by lumbar puncture also confirm presence of virusThe CSF rarely yields virus except in severe or fatal casesbut in full blooming JE infection serum antibody levelraises up to 4-fold whose measurement is only possible byCSF analysis Besides this a complete blood count (CBC)often helps to detect leukocytosis leucopenia anemia andthrombocytopenia in JE patients [35] (Table 1) Howeversequential changes in serum cytokines chemokines work asgood biomarkers for JE virus [36] There were increasedlevels of proinflammatory and anti-inflammatory cytokinesand a chemokine (monocyte chemoattractant protein-1) inthe serum of rats after JEV infection compared to controls[36] However significant alteration levels of cytokines andchemokine peaked at 10 dpi and declined significantly by20 dpi which shows neurological invasion in the acute stageof disease and partial recovery thereafter [36]

4 Plasma Biomarkers

Cells infected by viruses express viral antigens on theirmembranes long before the viral assembly takes place Ifa CTL and a suitable antibody are being made availablewith supporting active complement proteins it destroyslarge population of virus It is a very effective mechanism


which helps in mass destruction of virus-infected cells byusing antibodies (antibody-dependent cell mediated cyto-toxicity ADCC system) classical pathway of complementactivation phagocytosis and cytotoxicity mediated by CD8+lymphocytes Thus formation of antigen-antibody complexstimulates the Fc receptor on macrophages (CTLs) that helpsin viral clearance and evokes heavy complement mediatedcell lysis of virus or virus infected cells [37] However inresponse to a virus attack complement induces synthesis ofproinflammatory peptides (3a and 5ca) which help to reuniteand activate monocytes and granulocytes to the inflamma-tory site Further proteolytic fragments C3 (C3b C3bi C3dand C3dg) promote uptake by cells that express complementreceptor [38] In this process C3 convertase enzyme helpsand catalyzes the reactionThus after amplification hundredsof C3b molecules are generated which bind to nearby cellsand mediate damage to healthy cells by opsonization tophagocytic cells [39] C3b receptors help to form membraneattack complexThere is another possibility that C3 fragmentenhance viral antigen uptake facilitate antigen presentationby macrophages and DC and induces specific antibodyproduction andT cells proliferation [40]More specially C3bC4b and C3bi fragments play important role in opsonizationof antigen while C3b and C5b-9 help in neutralization ofvirus with C3b C5a and C5b67 causing extravasations andchemotaxis of neutrophils and monocytes [41]

In addition a number of cytokines play a significantrole in the development of an acute or chronic inflam-matory response and IL-1 IL-6 TNF-120572 IL-12 and manychemokines exhibit redundant and pleiotropic effects thatwork together and contribute to the inflammatory response[42] The inflammatory response provides early protectionfollowing infection or tissue injury by restricting the tis-sue damage to the affected site The acute inflammatoryresponse involves both localized and systemic responsesSimilar to JE Dengue virus flavivirus also show pathogenesisby chemokines and cause severity of infection as virusassociates to chemokine receptors CCR1 CCR2 and CCR4[43] Dengue virus infection also induces clinical symptomsrelated to tissue damage like thrombocytopenia hemocon-centration lymphopenia increased levels of transaminasesand proinflammatory cytokines It also shows antibody-dependent enhancement of growth in human monocytesthat is a serious risk factor in hemorrhagic fever [44]Besides this adhesion mechanisms regulate the migration ofmonocytes [45] and disease severity increases with systemicinflammation and activation of chemokine receptors whichplay discrete roles in the pathogenesis Besides chemokinesa variety of other mediators released by cells of the innateand acquired immunity also trigger the inflammation Otherdisease-causing viruses like JE virus may follow the sameprocess in primary infection and in generation of pathogen-esis Similarly in vaccinated population levels of IL-6 IL-8MCP(1) (monocyte chemo-attractant protein) MIP-1a andMIP 1b (macrophage inflammatory protein) were found tobe significantly higher which also play important role in thecellular immune responses to JE There is another possibilitythat JE virus inhibits the formation of human monocyte-derived macrophages to chalk out their phagocytic function

[46] Thus a virus-generated biomolecule during primaryinfection may become a strong biomarker by recognizing itslevel in control and early-infected patient [47]

Plasma contains immunoglobulins enzymes lysozymeand properdin a large protein All these plasma proteinsserve to destroy microorganisms including viruses and toxicsubstances that may enter into the blood from outside orfrom body tissues However mast cells continuously releaseheparin a conjugated polysaccharide that serves to preventcoagulation of blood during its circulation Albumins occurin plasma are mainly responsible for osmo-regulation incells and tissue fluids Similarly various ions viz chlo-rides carbonates phosphates sulphates and iodides of cal-cium magnesium and potassium maintain electrolytic func-tions Plasma also contains four interconnected mediator-producing systems which act through activation of G-protein-coupled constitutive or inducible receptors linked tosignaling pathways involving increased intracellular Ca(++)concentrations andor release of mediators including arachi-donic acid metabolites These important systems are kininsystem the clotting system the fibrinolytic system andthe complement system More specifically Hageman factor(factor XII) performs an intermediate function in the firstthree systems commonly when excessive damage occurs inthe tissues These four systems activate to form a web ofinteracting processes that generate a number of mediatorsin inflammation and have great pharmacological role Bothplasma and tissue kallikrein-kinin system work together andmaintain pharmacological properties while kinin receptorsand drugs reported to interfere with their actions By main-taining unique inter-relationship these mediators inducesmooth muscle contraction and increase vascular permeabil-ityC3a C5a andC5b67 and act together to inducemonocytesand neutrophils to adhere to vascular endothelial cells tomaintain extravasasive activity through endothelial lining ofthe capillary and migrate toward the site of complementactivation in the tissues [48] Activation of complementsystem results in influxes of fluid that carry antibody andphagocytic cells to the site of antigen entry [49] Some lipidsmainly phospholipids also act as inflammatory mediatorswhich include thromboxanes prostaglandins leukotrienesand platelet activating factors [42] Followingmembrane per-turbations phospholipids are degraded into arachidonic acidand lysoplatelet activating factor when subsequently con-verted into platelet activating factor that causes platelet acti-vation and induce inflammatory effects changing eosinophilchemotaxis and the activation of granulation of neutrophilsand eosinophils Acute phase proteins and other systemicresponses also play an important role in inflammation [50]However its elevated concentration if measured by ELISA inserum could be used as a strong biomarker

In addition plasma also contains prothrombin and fib-rinogen which help in blood clotting Moreover a kininsystem is an enzymatic cascade that begins when a plasmaclotting factor called Hageman factor (factor XII) is activatedfollowing tissue injury This activated Hageman factor acti-vates prekallikrein to form kallikrein which cleaves kinino-gen to produce bradykinin which increases vascular per-meability causes vasodilatation induces pain and triggers


contraction of smooth muscles Kallikrein also acts directlyon the complement system by cleaving C5 into C5a andC5b Another enzyme cascade that is triggered by damage toblood vessels yields less quantity of thrombin [51] Howeverinitiation of inflammation response also triggers less clottingsystem and yields fibringeneratedmediators of inflammationMore specifically the fibrino-peptides act as inflammatorymediators and induce increased vascular permeability andneutrophil chemotaxis Activated platelets release CD4OLwhich leads to increased production of proinflammatorycytokines IL-6 and IL-8 and increased expression of adhesionmolecules The integrin CD11bCD18 (MAC-1) also playsimportant role in clotting and binds two components ofthe clotting system factor X and fibrinogen Binding offactor X and CD11bCD18 increases the activity of factorX thereby promoting coagulation The fibrinolytic systemyields plasmin-generated mediators of inflammation thatinitiates the inflammation response by activating the classicalcomponent pathway Anaphylotoxins thus produced fromcomplement system bind to mast cell membrane receptorsand induce de-granulation after release of histamine andother pharmacologically active mediators These factors alsoact as opsonins and chemotactic molecules for neutrophilsand monocytes However several types of mediators play arole in the inflammatory response in which chemokines actas chemoattractants and activatemolecules during extravasa-tions

Nevertheless antibody and complement that play a rolein host defense against viruses are often crucial in containingviral spread during acute infection However more or less allenveloped viruses are susceptible to complement mediatedlysis The viral envelope is largely derived from the plasmamembrane of infected host cells and is therefore susceptibleto pore formation for the membrane attack complex Themembrane attack complex formed by complement activa-tion can lyses viruses and infected cells In addition JEVantigens associate with complement binding receptors andcell adhesion molecules present on the surface of neutrophilsaccurately detect cellular invasion of virus on neuronal cellsde-granulation and B cell phagocytosis [52] Further cleavageproducts of complement components C3a and C5a are calledanaphylatoxins which can be used as biomarkers if theybind to receptors on mast cells and blood basophils anddegranulation

Furthermore complement system also mediates neutral-ization of viral infection by forming larger viral aggregatesIt is also supported by antibodies when forming a thickcoat around virus particle that neutralizes viral infectivity byblocking attachment to susceptible host cells If the depositsof antibody and complement on virus particles are detectedcan give accurate results and become an important biomarkerin JE disease It also facilitates binding of the virus particles tocells possessing Fc or type 11015840 complement receptors Viruseshave developed a number of different strategies for evasion ofmembrane complex attack and natural immunity [53] Thesefoil the potential MAC activity by making interference withthe binding of complement to antibody-antigen complexesmimicking mammalian complement receptors in the virionHowever interactions of complement proteins to the virus

and host immune cells can sketch status of localization ofvirus generated tissue damage in SEV and other regionswith electron micrography which potentially help to detectpathological signs Further presence of JEV cell adhesionmolecules (CAMs) and virus specific binding can also workas JEV marker

However radio labeling techniques allow sensitive detec-tion of antigens or antibodies and other inflammatorymolecules (Table 1) Moreover inflammatory cytokinesreleased in response to infection can be measured by radiolabeling methods both inside body fluids and inside the hostbody cells However determining the levels of inflamma-tory cytokines mainly TNF-120572 can express upregulation ofinflammatory cytokines [54] Moreover number of CTLsformed and secretion of IFN 120574 from CD8 cells and bindingits to NK cells induces lytic activity [55] Similarly biotinlabels facilitate detection of small amounts of proteins byELISA or ELISOT Two-photon microscopy is also foundcapable of optically sectioning the material under exami-nation without causing phototoxic damage This techniqueallows the tracking of cells in their biological environmentovertime providing a temporal view of the behavior oflymphocytes following manipulation of the immune systemFurthermore green florescent proteins and their derivativesare used to analyze presence of living cells and dead cells intissues Similarly by using CFSI2 fluorochrome 56-carboxyfluoresciin diacetate succenyl ester techniques labeling ofimportant viral proteins in tissue and cells become possibleSimilarly labeling of antibodies with biotin and avidinallows accurate determination of the level of antibodyresponse during disease and nondisease state [56] Allthese are emerging biomarkers which help to establish thevirus-generated effects in human and animal hosts

5 Imaging Biomarkers

Neuroimaging constitutes an important component in thediagnosis of the underlying infectious agents in the centralnervous system infection Many new biomarkers are devel-oped that involve imaging technology to display cellular andtissue injuries in the central nervous systemdiseases Imagingbiomarkers have many advantages as they focus on imagingof viral encephalitis including that caused by exotic andemerging viruses Imaging biomarkers are usually nonin-vasive and generate intuitive multidimensional results onboth qualitative and quantitative data If combinedwith othersources of information imaging biomarkers can providemore accurate structural effects of viruses in infected patientsto clinicians and diagnose more authentically encephalitissyndromes These are noninvasive and relatively comfortablefor patients

Magnetic resonance imaging (MRI) and computedtomography (CT) are noninvasive neuroimaging techniqueswhich are used for detection of bilateral thalamic lesions withhemorrhagic regions and other structural abnormalities inbasal ganglia putamen spinal cord and cerebellum (Table 1)However to identify JEV generated hyperintense lesions inthalamus cerebrum and cerebellum T2-weighted MRIs are


used [57] In addition electroencephalography (EEG) alsoreveals diffuse delta pattern with spikes theta waves andburst suppression in nerve cells (Table 1) These methodscould help to establish JE virus selective infection in theneurons causing of ultrastructural changes in associationwith viral replication in the cellular secretory system prin-cipally involving rough endoplasmic reticulum (RER) andGolgi apparatus [58] In the early phase of infection RERof infected neurons showed hypertrophic changes contain-ing assembling virions within its dilated cisternae In thelater stage the SER became cystic and degenerative due totransport of multiple virions from Golgi apparatus to RERcisternae which was later on released into the cytoplasmwithin coated vesicles for exocytosis [58] JE virus infection ini-tiates endoplasmic reticulum stress and an unfolded proteinresponse [59] In the late phase of infection host body showssome regenerative changes in membranous organelles [58]

Similarly diffusion-weighted imaging or diffusion tensorimaging is proved to be superior to conventional magneticresonance imaging for the detection of early signal abnor-malities in herpes simplex virus encephalitis but also inenterovirus 71 encephalitis and in West Nile encephalitisMore specifically diffusion signals capture micro-structuralproperties of brain white matter but it is not feasible bystructural MRI scans However pattern of diffusion-weighedimaging signal changes in endemic diseases such as WestNile encephalitis Murray Valley encephalitis enterovirusand Japanese encephalitis is a newly emerging biomarkerHowever apparent diffusion coefficient ratios obtained bydiffusion-weighted imaging confirmpatients withHIV infec-tion [60] Similarly surface-enhanced Raman scatteringSERS can deliver chemical and structural information fromanalytes rapidly and nondestructively in a label-free mannerAlternatively SERS labels or nanotags when conjugatedto target-specific ligands can be employed for the selec-tive detection and localization of the corresponding targetmolecule It may have wider application in neuroimaging ofCNS disease viruses like JE [61]

Other imaging techniques used are magnetic resonanceimaging (MRI) optical coherence tomography (OCT) nearinfrared spectroscopy radio labeled fludeoxyglucose posi-tion emission tomography (PET) and diffusion tensor imag-ing which more exceptionally detect any type of atrophy thatoccurred in temporal lobes cerebral cortex thalamus andbrain stem In addition structural changes neurophysiolog-ical impairments are also determined by measuring take-upglucose in the body cells However tracking glucose sites ofinflammation can easily be explored because macrophagesmaintain high levels of take-up glucose However highutilization of glucose in the state of tumor growth or duringcellular necrosis that occurred due to invasion of viral toxinscould be explored by using imaging strategy MRI provideshigh spatial resolution and is very adept at morphologicalimaging and functional imaging MRI shows a sensitivityrange from 10minus3molL to 10minus5molL that is very limit-ing However for achieving molecular imaging of diseasebiomarkers using MRI targeted MRI contrast agents withhigh specificity and high relaxivity (sensitivity) are required

For this purpose commonly peptides antibodies or smallligands and small protein domains such asHER-2 affibodieshave been applied to achieve targeted imaging Functionalimaging help in measurement of acetylcholinesterase (AchE)and butylcholinestrase activities nicotinicmuscarinic recep-tor binding vesicular acetylcholine transporter and behav-iors and action of neuromodulators in undismayed anddiseased individuals These enzyme-based biomarkers aresuggested to be more sensitive and may help to decide earlystructural changes in brain of JE infected patients [62]

6 Anatomical Markers

JE is a severe neurological disease which causes high fatalityin infant groups Virus generates neurotrophic effects thatresult in encephalitis syndrome or acute susceptibility to CNS[63] JE virus mainly targets cerebellar Purkinje cells andcauses neurological signs such as ataxia [63 64] It also targetNCPs pools [65 66] inhibit cell growth proliferation [67] andcycle progression [68] It starts by invasion and destructionof immune cells by cytolytic mechanism which mostly targetNCPs pools [69] It results in a large number of neuronal celldeaths which occur due to microglial activation and robustinflammatory attack made by JEV virus [70 71] Howeverwithout knowing structural modifications in neuronal vas-cular andmuscular deformities due to JEV invasion it is verydifficult to find more appropriate authentic and suggestiveoutcomes for therapeutics and of JE vaccination Besidesthis serological findings are sometimes confusing becauseof high degree of cross-reactivity amongst the flavivirusantigens More exceptionally anatomical markers of JEVgenerated changes in thalamus substantia nigra brain stemhippocampus cerebral cortex cerebrum and spinal cord[72] With the help of biomarkers invasion of nerve cellsvirus transfer from blood stream to brain and occurrence oftransient viremia could also be identifiedHowever immuno-histochemical methods clearly display neurophysiologicalchanges in Japanese encephalitis patients [73]

JEV is a neurotropic virus that also targets developingCNS and infects embryonic NPCs and replicates inhibitingtheir growth and cell differentiation [67] JEV infection leadsto massive neuronal cell death [74] (Misrha and Basu) andcauses severe neuropathogenesis [75] JEV infected cells showextended lag phase during growth [76 77] JEV decreasesthe number of colony forming NPCs (NPCs pools) andreduce their self-renewal capacity and proliferative ability[68 69] A high necrotic cell death was observed in JEVinfected NPCs in comparison to control [69] JE virus alsocauses morphological changes in NPCs cells in infectedpatients and animal models which are localized by usingnestin as a marker in double immunohistochemistry [69]However nestin positive cells in the JEV infected brainlocalize two kinds of cells that is oval shaped cells and roundcells Round cells are specific markers of virus infestationMoreover FACS analysis of BrdU incorporated cells show asignificant decrement in its counts in cells from JEV infectedsubventricular zone of brain [69]


However as neuronal stem cells are self-renewable [78]but JEV infection shows aberrant formation of neurospheresin NPCs with progressive infection [69] Just after cellularinvasion primary NPCs form secondary neurospheres whichare identified by clonogenic assay [79] Neurosphere are free-floating eight-to-ten cell aggregates and their size becomesmaller in case of JEV infection However a more significantdecrease in the number of spheres generated from NPCs ofJEV infected subventricular zone than controls in vivo assay[69] In addition the apoptotic population of nerve cells isdetected by YUNEL assay Both flow cytometry of Annexin Vpropidium iodide stained NPCs and TUNEL assay revealedhigh number of apoptic cells with disrupted membrane tomorphological alterations and necrosis are other confirma-tory points for JEV invasion BrdU incorporation in vitroassay proved that JEV infection inhibits DNA synthesis andcell cycle progression in NPCs Further cell cycle kinetics inNPCs is analyzed by labeling with 7-ADD [80] a florescentDNA binding dye Among JEV infected cells most of thecells are found in G0G1 phase of cell cycle [81] which isan important cellular marker It indicates that JEV infectionblocks cell cycle progression through S phase inNPCs It alsoleads to up-regulation of GgtS phase checkpoint proteins [69]

Interaction of dendritic cells (DCs) with innate lym-phocytes (NK and NKT) represents a crucial event duringantiviral innate immune response Similarly IL2-activatedCD56(+) lymphocytes mediated immunomodulation andTNF120572 generate anti-viral effects during direct cell-to-cellcontact [82] However modulation of cross-presentation ofexogenous antigens through TLR signaling plays importantrole in anti-viral immune responses against JEV infection andmay help in development of effective vaccination strategy inthe future [83 84] Similarly ICAM-1 (intercellular adhesionmolecules) enhanced T-cell receptor signaling and activatedTh1 immune responses in the JEVmodel systemby increasingthe induction of CD4(+) Th1 cell subset and activatingdendritic cells [85] Further gene expression is affected byTNF120572 and IL-1120573 produced by JEV-infected microglia duringthe course of infection Before invasion virus attaches to hostmicroglial cells with a high affinity to laminin receptor pro-tein and nucleolin which are potential JEV binding proteins[86] By assaying the antibody inhibition of infection bothantilaminin receptor and anti-CD4 antibodies significantlyreduced virus entry [87] It acts as a strong biomarkerthat indicates involvement of multiple receptor protein thatmediate the entry of JEV to microglial cells with CD4 havinga major role in it

JEV is a neurotropic remerging virus that mainly targetsneurons enters microglial cells a neuronal cell type [86]and causes massive neuronal destructiondysfunction [87]Virus attacks neuronal cells causing high inflammationin the CNS and try to impair functional and structuralintegrity of BBB and other regions of brain BBB (blood-brain barrier) preempts the damage to CNS from exogenousmoleculesmainly virus generated toxins Similarly astrocytesplay key role in regulation of inflammation and homeostaticmaintenance of the central nervous system [88] (Mishra etal 2007) JEV infection increases the expression of astrocytespecific glial fibrillary acidic protein (GFAP) Successively

after JEV infection both nerve growth factor (NGF) andcellular neutrophin factor (CNTF) are also elevated whichprevent ROSmediated neuronal cell death in JE infected host[88] If the protective efficacy of astrocytes to JE is amplifiedit will modulate the adaptive response of the brain to inducehigher neuroprotection [88]

Severe dengue virus (DENV) disease is associated withextensive immune activation characterized by a cytokinestorm However previously elevated levels of lipopolysac-charide (LPS) in dengue virus patients are found to becorrelate with clinical disease severity [89] Similarly JE viruscaused severe neuroinflammation which start with robustexpression of proinflammatory cytokines and chemokineswith increased number of infiltrating inflammatory cells intothe brain The virus mainly infects neuronal cells and causesan inflammatory response after invasion of the parenchymaof the brain Histopathology confirms the infiltration ofleucocytes and there was a marked upregulation in theexpression of genes relevant to infiltration It is also associatedwith involvement of monocyte and macrophage receptorCLEC5A in severe inflammatory response in JEV infectionof the brain However expression level and molecules ofneuroinflammation can work as important biomarkers fordevelopment of appropriate future diagnostic tools for JEtherapeutics and prophylactics [90] The death of neuronsis frequently observed in demyelinated axons which isambiguous However presence of myelin-specific antibodiesin sera in mice with evident symptoms shows presence ofvirus in neuronal cells Further it is strengthen by specificT cells proliferating in response to stimulation by myelinbasic protein (MBP) in mice It shows autoimmunity thatmay play an important role in the destruction of componentsfor example MBP of axon-surrounding myelin resulting indemyelination in the mouse brain after infection with the JEvirus [91]

Minocycline a semisynthetic tetracycline has been foundto be broadly protective in neurological disease It mainlyshows neuroprotective role and slow down inflammationand cell death in experimental models [92] However abreakdown in BBB is detected by finding leakage of protein-bound Evanrsquos blue dye into the brain tissue of experimentalanimal Semiquantitative RT-PCR revealed an upregulationof chemokine receptors and adhesion molecules followingJEV infection Immunostaining showed leukocyte and neu-trophil infiltration following JEV infection Intraperitonealinjection of minocycline beginning 24 h after-JEV infectionabrogated the effects by reducing BBB damage decreasingexpression of iNOS Cox-2 and VEGF and also by reducingthe elevated level of transcript of chemokine receptors andadhesion molecules in the brain Matrix metalloproteinases(MMPs) are known to disrupt the BBB and minocyclinewas found to significantly decrease the activity of MMP-9in brain tissue homogenates and appears to maintain blood-brain barrier integrity following JEV infection [92]

JE virus infection evokes acute encephalopathy in chil-dren a clinical syndrome with high mortality and neuro-logical sequelae [47] Virus invasion in patients generatesymptoms such as impaired consciousness and convulsive


status epilepticus with hyperpyrexia [93] It is character-ized by detection of biphasic seizures and late reduceddiffusion (ASED) in MRI tests [93] However there is nospecific biomarker for early diagnosis of acute encephalopa-thy syndrome available but tau protein and 8-hydroxy-21015840-deoxyguanosine (8-OHdG) seem to be potential biomarkersBiomarkers of patients with acute encephalitis and acuteencephalopathy are screened in blood and serum [35] More-over CSF PCR is highly useful to detect acute encephalitisin patients Mainly biomarkers for brain injury are con-sidered through a systematic screening of most vital andfunctional cells and tissues which are affected by neonatalencephalopathy [35] However different hosts show differentviral tropisms and host immune responses prior to viral entryinto the central nervous system It is possible that it dependson antigen profile and protective strength of immune systemOther than damaging cells and tissues JE virus affect theglutamate aspartate transporter glutamate transporter 1 andceruloplasmin levels There was observed an elevated level ofLDH in the animals infested with wild strains of viruses [94]

Further detection of N-glycosylation sites of JEV virus Eprotein and focal pathological effects such as focal neuronaldegeneration with diffuse and focal microglial prolifera-tion and lymphocyte perivascular cuffing strongly work asbiomarker Brain microvascular endothelial cells representa functional barrier and could play an important role inleukocyte central nervous system trafficking [95] Upon entryand infection of the CNS these viruses can induce a rapidinflammatory response characterized by the infiltration ofleukocytes into the brain parenchyma Both chemokines andtheir receptors are involved in coordinating complex leuko-cyte trafficking patterns that regulate viral pathogenesis invivo However key cellular events occur during the infectionprocess and the immunodiagnostic role of these cells willbecome a strong future biomarker to identify the infiltratingvirus in host cells [96]

7 Physiological Markers

Though it is very difficult to detect metabolic impairments inpathological cells cell cultures of neuronal cellsnerve cellsa very difficult task If are possible these can be used forjustifying virus generated physiological defects It would alsohelp to observe behavior of viral antigens in vitro to variousnerve cell membrane molecules neurotransmitters ions andsynaptic binding of inhibitory proteins Such cell culturesystems will certainly help to explore cellular entry of virusits invasion and progression of disease and pathogenesisHowever measurement of acetylcholine activities in infestedcells is still lacking More exceptionally the behavior ofvirus toxins to Na+K+ATPase pump in sensory and motornerve fibres and proton deficiency can be correlated withpathogenesis JEV infection caused increased intracellularROS production and activation of ASK1-ERKp38MAPK sig-naling in human promonocyte cells [97] Similarly increasedlevel of free radicals due to oxidation of bio-molecules inpatients body also indicates higher neuronal damageinjury[98] that works as an important clinical biomarker of

many viral infections [98] However maximum increasedlevels of ROS species (ROS) nitric oxide (NO) peroxinitite(OONO)(minus) causes apoptotic cell death of neuronal cellsIt also aids to generate acute JE with representative signsand symptoms of neuronal shrinkage and tissue necrosis[100] Further downregulation of thioredoxin increasedintracellular ROS and activation of ASK1ERKp38 MAPKsignaling are associated with JEV induced apoptosis [97](Yang et al) However thioredoxin prohibits JE pathogenesisby suppressing oxidative stress pathway [97] Further viralinfection inhibited the expression of cell maturation surfacemarkers (CD40 CD80 and CD83) and MHCl and impairedthe ability of P3-infected DCs for activating allogenic naıvecells [100] It impairs T-cell maturation modulates cytokineproductions and expanded regulatory T cells [97] Howeverboth structural and functional impairments occurs in neu-rons of infected patients if identified exactly could be used asimportant biomarkers to know virus generated pathogenesis

However other than damaging cells and tissues JEvirus affect the glutamate aspartate transporter glutamatetransporter 1 and ceruloplasmin levels There was observeda rapid increase in total LDH level in the animals infestedwith wild strains of viruses that indicates severity of virusinfection [94] However mice infected with wild strains of JEshowed all five isoenzymes among which LDH 1 disappearedafter 12 days of infection but LDH 2 and LDH 3 persistedfor 3 week while WN virus stain showed an extra band nearLDH 4 [94] Contrary to this attenuated strain of JE did notproduce any change either of the total content of the enzymeor of the isoenzyme pattern In the plasma of mice infectedwith wild strains of WN viruses only 4 isoenzyme bands(LDH 2 3 4 5) were detected in the gel More important isLDH 3 persisted longer than LDH 2 Similarly an alterationin serum sodium level liver enzyme function and ADHsecretion also mark JEV generated morbidity Further anelevation in aspartate aminotransferase (AST) and alanineaminotransferase (AMT) enzyme levels indicate virus inoc-ulation in patients [101] though it cannot identify severityof Japanese encephalitis or its outcome Similarly almost noreport is available on hormonal changes in JE patients thosewho have recovered Certainly structural changes occur inendocrine glands hormone deficiency and hypersecretionsmay correlate to the JEV generated effects However aninstant increase in cellular enzyme mainly LDH and glucosetransporters and hormonal level could work as an importantdisease marker for JEV diagnosis in future Hence metabolicimpairments are correlated to glucose utilization then theseverity of disease resulting in neurophysiological changescould be gauzed [62]

8 Immunohistochemical Biomarkers

Similarly presence of viral antigens in tissue and cellsalso unravel neuropathogenesis caused by JEV Howeverimmunocytochemical localization of viral antigens by usingimmunofluorescence and immunohistochemical methods[102] helps to know structural changes caused by JE virus andworks as an important biomarker (Table 1) Similarly flow


cytometry also helps to recognize viral invasion cell deathand disease prevalence inside host [69] However detectionof pathological changes by immunohistological methodsin thalamus substantia nigra brain stem hippocampuscerebellum and spinal cord by more clearly display thereasons of morbidity in JE patients Similarly pathologicalchanges such as focal neuronal degeneration with diffuseand focal microglial proliferation and lymphocytic perivas-cular cuffing could also clarify cellular interactions of virus-secreted molecules serving as biomarkers However afterestablishing correlation between structural changes and cel-lular and tissue specific abnormalities JE disease progressionand status could be decided Furthermore for detection ofJE progression and its related effects neurosphere culturesare used [69] Similarly for fast analysis of JE virus antigensan indirect IgG immunofluorescence test (IIFT) is applied(Table 1) It is highly sensitive and specific method [102]which is used to detect antigen titers in the mouse brainmainly in hemisphere by confirming the binding of JEVantigen with rabbit anti-JEV serum It is highly usefulmethod for diagnosis of acutely infected persons and is avaluable alternative to the other established methods fordetecting anti-JEV antibodies and humoral immune responseafter vaccination [6] However immunological relationshipsamong flaviviruses can be established by detecting the levelof MAbs in immunofluorescence and neutralization tests(Table 1) [103 104]

JE virus targets neurons and generate neurotropismthat persists in human cells mainly in nerve and differentblood cells (erythrocytes lymphocytes granulocytes andmonocytes) It is quite interesting that JEV could not replicatein erythrocytes granulocytes or lymphocytes but it culturesin monocytes because these cells support virus replication[105] but JEV could replicate more efficiently in neurob-lastoma (HTB-11) cells than in monocytes after infectionfor 48 h However JEV-infected neuroblastoma cells sufferedheavy cell apoptosis in 2 days and decreased viability to lessthan 1 in 5 days [105] while monocytes could take up JEVrapidly and display a log scale increase of intracellular JEVtiters in 9 h after infection that prolonged for more than 3weeks Thus JEV-infected monocytes play an important rolein harboring JEV for eventual transmission to NB cells andmodulation of JEV-induced NB cell apoptosis may be usefulin treating patientswith JE [105] It is confirmed by expressionof viral NS3 antigen and virus plaque-forming units

More specifically influenza virus is surrounded by anouter envelope a lipid bilayer acquired from the plasmamembrane of infected host cells during the process ofbudding Besides this two hemagglutinin (HA) and neu-raminidase (NA) proteins form radiating projections insertedinto the outer envelope More specifically hemagglutinintrimer binds to sialic acid groups on host cell glycoproteinsand glycolipids by way of a conserved amino acid sequencethat forms a small groove in hemagglutinin molecule [105]While neuraminidase cleaves N-acetylneuramic acid fromascent viral glycoproteins on host cell membrane and facil-itates viral budding from the infected host cells [105] Simi-larly glucosidase inhibitor of endoplasmic reticulum blocksthe trimming step of N-linked glycosylation and helps to

eliminate the production of several endoplasmic reticulum-budding viruses such as dengue type II (DEN-2) and JEV83

Serum proteome cytokines and inflammatory analysisof adults with primary dengue infection reveal predictivemarkers of DHFThese markers display three different stagesof infection representing the early febrile defervescenceand convalescent stages Using fluorescent bioplex assays27 cytokines were detected in serum samples of DHFinfected patients Additionally multiple mass spectrometryand comparative analysis of serum proteome as well asmeasurements of protein adducts-3-nitrotyrosine and 3-chlorotyrosine as surrogate measures of free radical activityact as molecular marker for DHF [106] Few immunologicalstudies provide evidence that TLR2-MyD88 and p38 MAPKsignal pathwaymight be involved in JEV-mediated inhibitionof cross-presentation of soluble and cell-associated antigensHowever modulation of cross-presentation of exogenousantigens through TLR signaling has important implicationsfor antiviral immune responses against JEV infection It willhelp in will development of effective vaccination strategies[84]

In addition the production of reactive oxygen speciesproduction and activation of ASK1-p38 MAPK signalingpathway might be associated with JEV NS2B-NS3 pro-tease induced mitochondria It mediate apoptosis in humanmedulloblastoma cells and serve as an important biomarkerfor JEV In addition it might be useful in recognition ofcellular and molecular pathogenic effects induced by JE virusinfection [97] It clearly shows that fatality of infected patientoccurs due to extensive neuronal dysfunction rather thanneuronal destruction in the CNS [58] Further oxidativedamage also plays an important role in the pathogenesisof viral infections of the nervous system [98] Further forimmunocytochemical localization of virus proteins (NS3)and cloning expression of NS3 genes [75] allow to detectneurovirulence generated by nonstructural JE virus pro-teins [75] Similarly western blot and immune-florescenceanalysis using the anti-NS3 antibody also explore effects ofnonstructural proteins in human and experimental animals[75] Thus double immunostained cells with the anti-NS3antibody and anti-flag antibody clearly show the presence ofvirus secreted antigens However virus structural proteinseither recombinant or natural help in establishing the causeof infection and are considered as important protein markersof JE virus Further oxidative damage also plays an importantrole in the pathogenesis of viral infections of the nervoussystem [98]

9 Virus Proteins as Biomarkers

JEV contains positive single stranded RNA genome approx-imately 11 kb in length Virus genome contains a single openreading frame with a well-arranged gene order as 51015840 C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5 31015840 [107108] It encodes viral proteins [109 110] mainly a precursorpolyprotein having three structural proteins (C prM andE) and seven nonstructural proteins (NSI NS2 NS2B NS3NS4A NS4B and NS5) [111] Among nonstructural proteins


NS3 is a multifunctional protein having 619 amino acidresidues and shows enzymatic activities like serine proteasehelicase and nucleoside triphosphatase NS3 plays importantrole in the processing of the viral precursor poly protein andthe replication of viral genomic RNA [112] In infected cellsNS3 is associated with microtubules and tumor susceptibilitygene 101 protein and plays essential role in viral packingintracellular trafficking of various viral components It wasdetected in the brain of JEV infected patient mainly inthe cytoplasm of pyramidal neurons of the cerebrum [113]granule cells small cells and Purkinje cells of the cerebellumafter 12 h after-infection The Purkinje cell of the cerebellumis one of the target cells of JEV infection [9] NS3 is animportant agent that generates neurovirulence in patients[114] Suppressive effects neurovirulence and host immuneresponses generated by different JE viral proteins and anti-gens are mentioned in Table 1

The flavivirus nonstructural glycoproteinNS1 is a cell sur-face protein (soluble entity) that generates neurovirulence inhost neural cells after peripheral inoculation of virus amulti-functional protein that shows mechanistic function [115] andassists the virus in neuronal invasion [116] It plays importantrole in pathogenesis and cellular profusion and acts as avirulence determinant [117ndash119] and serves as a marker ofDengue virus infection in man [120] and mosquitoes [121]Both secreted and cell-surface-associated NS1 are highlyimmunogenic and implicate disease pathogenesis [122ndash126]This also occurs as a viral antigen and circulates in thesera of JE infected patients It shows host immune responseand elicits protective immunity in mice [127] It plays animportant role in establishment of pathogenesis and is usedto generate protective antibodies against flavivirus [128ndash130] It shows homology with dengue virus protein and itsdeformation affect CNS in mouse [131 132] Intracellular NS1plays an essential cofactor role in virus replication and hasbeen shown to colocalize with dsRNA and other componentsof replication complexes [133 134] It was found at differentcellular locations either cell-membrane-associated (mNS1)in vesicular compartments within the cell or on the cellsurface and as a secreted lipid-rich extracellular (nonvirion)species (sNS1) [135 136] or hexameric lipoparticle [137ndash139] More specifically secreted form of NS1 shows widerinteractions with host proteins and other bio-molecules andfound implicated in immune evasion strategies and playinga direct role in pathogenesis Therefore NS1 functions asan important biomarker for early diagnosis of JE diseasein infected hosts However interaction of NS1 related to itsstructure and trafficking within and from the infected celland its possible role in viral replication may have very highvalue in diagnostic applications

In addition two more structural proteins C and prM arealso identified in JE virus by using cDNA analysis Theseproteins contain glycosylation sites that show similarity withTBEV and WNV N-linked glycosylation site in prM or Eprotein and display protective potential More specifically inJE viruses the prM protein contains one putative N-linkedglycosylation site at N15E protein and another site at N154 Ifdeletion occurs in the above site it lead to a decrease in viralrelease [140ndash142] Similarly mutations occurred in envelope

and coat protein work as real elements of neurovirulencedeterminants in mice It is proved by preparation and useof chimeric viruses [143] For example poliovirus infectionis largely confined to a specific subpopulation of neuronalcells occur in human central nervous system and showsPV tropism and neurovirulence [144] However mutationsgenerated in putative N-linked glycosylation sites in Japaneseencephalitis virus premembrane (prM) and envelope pro-tein(E) showed enhancement in protective potential [145]Similarly N-linked glycans of viral proteins play importantrole inmodulating immune response in host cells [146]Theseare also important formaintaining appropriate antigenic con-formations mainly neutralization epitopes that potentiallyalter the proteolytic susceptibility of these proteins [146 147]

There is another major structural envelope protein Ethat contains numerous neutralization epitopes which playimportant role in viral attachment membrane fusion andentry of virus into host cell E protein also contain oneputative N-linked glycosylation site at NS154 [132] that playsmajor role in determination of virulence phenotype Itsputative receptor binding domains induces the host immuneresponse [148 149] E protein showed single amino acidsubstitutions which are sufficient to cause loss of neurovir-ulence [150 151] Besides E (envelope) protein NS3 is themain protein that is responsible for pathogenesis and showimmune response However viral proteins NS3 proteasein association of NS2B cofactor significantly induce higherdegrees of apoptosis and trigger higher caspase 3 activitiesin human medulloblastoma cells [64] Similar to NS1 viralproteins E proteins are well-known targets of the protectiveantibody response against flavivirus infection and containvirulence determinants [128ndash130] (Table 1)

However so many biological markers of neurovirulencehave been identified [152] but molecular determinants ofvirus specific factors which account for virulence are stillunknownThesemight be highly specific more confirmatorythan biological markers because they are encoded with inmultiple region of neurotropic RNA viruses [153ndash155] It isalso proved by single site mutations generated in the fla-vivirus genome which encode envelope protein hinge regionthat resulted in a significant increase in virulence in miceand monkeys [156] Mutations that occur in structural andnonstructural viral proteins are responsible for generationof neurovirulence These might be of reversion or deletiontype Similarly mutations occurred in matrix (M) proteinof vesicular stomatitis virus (VSV) generate neurovirulenceThese M protein mutants of VSV can be used as vaccinevectors [157] Interestingly measles virus also shows veryhigh neuroinvasivness in animal models but shows limitingneurovirulence in humans Similarly poliovirus causes veryhigh susceptibility to CNS and grows in neural cells butshows a limited neurovirulence in host Furthermore serialpassages done for yellow fever virus (YF 17D) in mousebrain enhances neurovirulence and causes a reduction insurvival time after intracerebral inoculation of experimentalmice [158] However severity of infection increases neurovir-ulence which may occur due to virus invasion in nervecells mainly in brain It also shows inflection of virus inspreading route mainly in neuroaxis brain and spinal cord


[159] More exceptionally an adjacent stem loop structuresidentified within the JRES and internal ribosomal entry sitecooperatively determine neuropathogenicity [144] Howeverweaker interactions occurred between virus antigens andhost immune cells generate chances of more neuronal inva-sion by virus that may lead to high pathogenesis [160]In such a condition both cellular and humoral responsescannot strike well upon virus and generated molecules andeven the bodyrsquos own infected cells [161] However candidatemolecules which are secreted after invasion of virus by hostbody cells can be used as goodbiomarkers for neurovirulencedetermination [162]

Recently microarray researches cleared that neurons canmake their own defense against Japanese encephalitis viralinfection even that they do not show power of regenerationIt is a very challenging job because neurons are immuno-logically quiescent and an improvement in proinflammatoryeffects is very difficult task for immune-mediated controlof viral infection and repairing of neuronal injury If it willbecome possible to have some novel inducers of neuronalregeneration it will be a land mark step for developingstrategies for limiting the severity of CNSdiseasemainly paininflammation and neurological impairments in patientsHowever monocyte and macrophage receptor CLEC5A theyare found involved in severe inflammatory response in JEVinfection could be sloweddown

Similarly identification of different protein functionsof structural and nonstructural proteins of JEV genomemay also facilitate a mechanistic understanding of Japaneseencephalitis virus (JEV) infection However protein func-tions common to both structural and nonstructural proteinssuch as iron-binding metal-binding lipid-binding copper-binding transmembrane outer membrane channelsporespore-forming toxins (proteins and peptides) could workas important biomarkers Similarly nonstructural proteinsperform functions like actin binding zinc-binding calcium-binding hydrolases carbon-oxygen lyases P-type ATPaseproteins belonging to major facilitator family (MFS) secret-ing main terminal branch (MTB) family phosphotransfer-driven group translocators and ATP-binding cassette (ABC)family group of proteins could also establish diagnostic factsabout JEV and are considered as important future biomark-ers Most flavivirus nonstructural (NS) proteins correlatewith virus-induced inflammation and immune escape How-ever structural proteins besides belonging to same structuralgroup of proteins (capsid structural and envelope) they alsoperform functions like nuclear receptor antibiotic resistanceRNA-binding DNA-binding magnesium-binding isom-erase (intramolecular) oxidoreductase and participate intype II (general) secretory pathway (IISP) [163] It will alsohelp to develop new drugs

JEV protein E shows a potential to induce antiviralresponses by synthesizing anti-JEV neutralization antibodiesHowever E protein derived peptides also contain virusneutralization epitopes which assist in generation of JEV-neutralizing antibodies [164] Similarly plasmid encodingJapanese encephalitis virus premembrane and envelop genesgenerate immune responses and induction of protective

immunity against Japanese encephalitis in mice [15] Follow-ing an immune response these also induce specific memoryB-cells and long lasting antibodies in animal hosts [165]Similarly membrane anchored and secretory envelope pro-teins elicit immune responses in experimental animals [14]However it is established that plasmid encoding Japaneseencephalitis virus protein induce neutralizing antibody orcytotoxic T-lymphocytes in mice [16 17] Further neutraliza-tion ligands selected from Phage displayed librarian mimicthe conformational epitope on domain III of the Japaneseencephalitis virus envelop protein [166] Similarly human Cvirus envelop protein E1 contains N-glycosylation sites andenhances specific cellular and humoral immune response[167]

More specifically interferon (IFN) antagonists ofJapanese encephalitis virus (JEV) proteins contribute tothe JE pathogenesis [168] However NS4A proteins ofWest Nile virus and dengue type 2 virus demonstratedinhibition of IFN signaling Similarly JEV NS4A withoutthe C-terminal 2 K domain partially blocks activation ofan IFN-stimulated response element (ISRE)-based cis-reporter by IFN-alphabeta It also significantly inhibits thephosphorylation levels of STAT1 and STAT2 but not TYK2in the IFN-treated cells [168] Moreover the N-terminus ofan RNA helicase DDX42 protein specifically binds to JEVNS4A in vitro and such interaction is localized in humanmedulloblastoma TE-671 cells by confocal microscopyImportantly the expression of N-terminal DDX42 is able toovercome JEV-induced antagonism of IFN responses [168]However the level of IFN alpha and beta works as importantdisease marker of JE virus infection

However chimeric yellow fever (YF) virusJapaneseencephalitis (JE) virus vaccine (ChimeriVax-JE) constructedby insertion of the prM-E genes from the attenuated JE virusSA14-14-2 vaccine strain into a full-length cDNA clone ofYF 17D virus [169] induces protective immunity against JEvirus after immunization Moreover single site mutationsdone at E279 position were located in a betasheet in thehinge region of the E protein that is responsible for a pH-dependent conformational change during virus penetrationfrom the endosome into the cytoplasm of the infected cell[169] After intracerebral inoculation the E279 Lys virus wasrestricted to extraneural replication in monkeys as viremiaand antibody levels were significantly reduced compared tothose for the E279 Met virus [169] More exceptionally itshows a reduced viscerotropism in humans in comparison tomice [169] However new antigenic sites generated by usingsite directed mutagenesis in virus genome and expression ofthese mutated virus genes could help to mark the negativeand positive effects of prMgenes in experimental animals andproved to be good biomarkers for identifying JEV generatedpathogenesis

Furthermore Japanese encephalitis virus (JEV) is alsodetected in clinical samples by using one step TaqMan reversetranscription polymerase chain reaction (RT-PCR) (Table 1)It is highly sensitive specific rapid and quantitative diagnos-tic method used for the fast detection of JEV in laboratoryand field collected samples [170] It helps in the quantificationof JEV which is accomplished by preparing a standard curve


plotting cycle threshold values (C(t)) versus infectivity titer[170] It is used for detection and quantification of JEVand its RNA genome in plasma samples in 2 days afterinoculation experimental animals with KV1899 strain [170]This test could also help to establish strain specific variants ofRNA genome which will work as perfect biomarkers at themolecular level for exploring alternate reasons of JE-inducedneuropathogenesis and disease

10 Molecular Markers

Certain biomarkers which can display role of genes in diseasepathogenesis and mutations evolved and its expressionprofiles could explore the disease status andmorbidity relatedeffects These biomarkers can be identified by using basicand acceptable techniques used in genomics and proteomicsof JE virus However genomic approaches such as northernblots SAGE DNA micro array are used to find disease-specific candidates related to gene structure and functionSimilarly regular protein profile obtained from an infectedand uninfected patient could be obtained by analyzing bodyfluid tissue and cells on 2D PAGE LC-MS SELDI-TOF or(MALDI-TOF) antibody microarray and tissue microarraycould establish changes related to structural and nonstruc-tural protein in human hosts Moreover all structural andnonstructural protein changes occur in patients at differentperiods could clearly establish morbidity caused by Japaneseencephalitis virus Further genome-specific markers help todetect JE virus mutations occurred in ecologically adaptedantibody resistant strains Other tests which are used fordetection of virus are reverse transcription PCR (RTPCR)and electron microarray which are also used to establishpresence of JEV in clinical samples (Table 1) Therefore fora quick start in treatment a confirmed diagnosis of JE basedon rapid immunodiagnostic tests is essentially required

Similarlymutation analysis of virus proteins and its inter-actions to disease marker genes could make JEV diagnosismuch easier However statistical analysis of the envelope geneand prM region of JEV virus could find significant variationsin nucleotide sequences Further presence of selective forcesacting on these regions investigated by computing the relativerate of synonymous substitutions could explore heteroge-neous genotypes circulating in endemic population [171] Itcould work as an important biomarker Estimates of meanof nucleotide distances for different region of the E genecould establish the divergence occurred in flavivirus and canpresent possible divergence in future strains However com-parative analysis of complete genome sequence and its full-length sequence based phylogenetic analysis could confirmthe particular JEV strain belonging to particular genotypeHowever molecular substitutions per site could explain therole of distantly placed viruses and its possible neuroviru-lence in human host If compared the polyprotein as a wholethen unique difference in amino acid substitutions could beachieved it will help to know the functional differences in anewly formed protein and its workable antigenicity Howevereither these old and new functional differences created ornatural one could suggest all the possible modifications in

the epitopes It will work as an strong confirmatory markerfor determination of level of morbidity in different JE virusstrains circulating in the endemic area DNAzyme mediatedinhibition of virus replication is an important molecularmarker to find presence of neurotropic virus titers inside hostThis oligonucleotide mediated inhibition also work as drugbiomarker for JEV [172]

Moreover a molecular analysis of transcriptomic data ofJE virus could ably find exact genotype and its generatedpathogenesis in human hosts Therefore cloned genes canbe transfected into cultured cells and examination of tissuespecific gene expression and its comparison in different cellsmay provide overall information at gene level functions andits alternations more accurately Further identification ofcandidate host gene and systematic mRNA profiling couldestablish real cause of JEV pathogenesis Moreover microar-ray analysis of mRNA expression profiles in spleen and braincould explore JEV infection and virus induced cellular andmolecular changes in experimental animals and human hosts[173] These circulating viral microRNAs have been reportedas potential biomarkers for the neuroinvasive diagnosis ofvirus infection [174] These are useful for diagnosis of viralinfections since viral microRNAs should be released in theextracellular space after the death of infected host cells [175]Interestingly viral microRNAs in body fluids varies fromhost to host may serve as specific markers for viral infectionand disease progression or for therapeutic monitoring anddrug development [175ndash177] It is well known that significantpathways involved in differentially expressing genes areinvolved in cytokine-cytokine receptor interactions naturalkiller cell mediated cytotoxicity antigen processing and pre-sentation MAPK signaling and toll-like receptor signalingHowever these could work as multidimensional biomarkerwhich canmake a clear picture of various biological processesand its related secretory molecules particularly comparing alarge data set fromDNAgene expressionmicro-array analysisfrom different JEV infected patients

However to emphasize the effects of individual virusgenes and cluster of genes its expression level couldstrengthen the role of multiple genes in establishment of JEdiseaseHowevermultivariate functional genomic data couldtell about time bound assimilation of new mutations andinduction of pathogenic features in different hosts thoughwhich attain wider neurovirulence However covarianceparameters of single andmultiple gene functions could estab-lish multiprocess pathways and variability across individualsIn addition analysis of time course of gene expression datacould explain temporal shifts in gene arrangement due tosubstitution mutations However most distinguished impactof gene on protein related variations and its best possiblehost responses will be known These may be used as strongmolecular biomarker for future

Other strong markers could be obtained from met-abolomics lipidomics glycomics and secretomics studiesThese are the most commonly used techniques whichidentify metabolic pathways involved during virus cycleinside host body mainly pathways followed for generation ofpathogenesis However utilization of metabolic componentscombustion and end product formation could help to assess


the disease status partially Further analysis of lipids pro-vides unique physical properties of certain lipids in diseasestate However improvements in new analytical platformshave made it possible to identify and to quantify most oflipids metabolites from a single sample However parametersrelated to carbohydrate lipid and protein profiling on massspectrometry chromatography and nuclear magnetic reso-nance can be used as a marker for JE diseases Mass spec-trometry is used to delineate the relative concentration andcomposition of high-density lipoproteins (HDL) particlesfrom lipid extracts isolated from JE patients and healthy vol-unteers could express some specific changes However levelsof HDL sphingomyelin phosphatidylcholine triglyceridesand cholesterol esters could assign involvement of somespecific pathway more suitable for virus invasion Similarlypresence of certain metabolites in blood and urine are well-known biomarkers for influenza virus and Staphylococcusaureus coinfection [178]

11 Therapeutic Markers

Therapeutic markers could establish specific interactionsof drugs to virus and host body molecules and cells Ifthese therapeutic effects are correlated pathways of diseaseoccurrence morbidity status and clinical care targets couldbe decided in JE patients However there is an utmost needof chemotherapeutic agents which could slow down thevirus multiplication and cell invasion and neurotropismThese should inhibit formation of virus structural and non-structural envelope proteins and can suppress the lethalaction of virus generated molecules Furthermore actionroute of plant origin anti-viral components and its inhibitoryeffects must be investigated on virus genome genes andproteins Moreover virus specific chemotherapeutic agentscould also manage inhibition of virus multiplication thatmay result in inhibition of infection at an earlier stageMoreover herbal therapeutic agents who can neutralizethe virus-generated effects and show sustainable neuropro-tective effect are to be essentially explored These novelmolecules should possess enough potential to decrease theviral load activate caspase-3 activity reactive oxygen andreactive nitrogen species microgliosis and proinflammatorycytokines in JE infected patients [179] Interestingly treat-ment with arctigenin improves the overall stresses caused byJEV and behavioral changes occurred in JE patients [179] Itshows antiviral neuroprotective anti-inflammatory and anti-oxidative effects and much successfully reduced the severityof disease induced by JEV [179] Similarly inhibition ofubiquitin-proteasome system by curcumin causes reductionin infective viral particle production from previously infectedneuroblastoma cells [180]Moreover bioorganic compoundswhich can significantly cut down virus generated cellularand tissue stress and injuries and might show repairingcapacity will be on high agenda More exceptionally thera-peutic molecules which can stop virus lethal challenge virusinvasion in neuronal cells and to diffuse the virus load inthe patient and show quick solubilizing anti-inflammatoryeffects and restore behavioral impairments in JE patients are

to be highly required and explored Minocycline is found tobe broadly protective in neurological disease which mainlyreduce inflammation cell death and abrogated the effectsby reducing blood-brain barrier damage [92] It significantlyreduces microglial activation inhibits caspase 3 inductionand viral replication following Japanese encephalitis [68]Similarly few lectinmolecules such as collectins ficolins [181]and selectins [182] play important role in generation of innatedefense acute phase proteins [46] that finish the infection[183] Similarly mild hypothermia therapy in children helpsto reduce brain edema [184]

In addition other strategies like RNA silencing andinterference activation of complement system are used toprotect from JE virus infection Similarly a short hairpinRNA or lipid complexed small interfering RNA (siRNA)is used for RNA interference before virus challenge theimmune system [185] It also suppresses fatal encephalitisgenerated by two different flaviviruses However use ofvarious types of cytokines complement proteins enzymesantibodies and passive transfer of activated CTLs T cells Bcells and NK cells can be used to destruct viral infectionFurther gases like nitric oxide (NO) have been shown tosuppress Japanese encephalitis virus (JEV) RNA synthesisviral protein accumulation and virus release from infectedcells [186 187]

Further to check the skipping and mutating behavior ofvirus cross-protective vaccines are to be prepared becausenew heterologous genotypes of JEV are emerging in endemicareas Hence new vaccines will be required to elicit protectivelevels of neutralizing antibodies against heterologous strainsof genotype I-IV [188] In addition novel viral antigens fromstructural proteins or its derived peptides may be used forpreparation of strong vaccines that can induce an overall anti-viral state by generating potential immune responses againstJE virus [189 190] Similarly a synthetic oligonucleotide-based DNAzyme significantly inhibit JEV virus replicationand proliferation of Japanese encephalitis virus mouse brainand in vitro cell culture [171] It also protects JEV-infectedmice from death It results in a sharp reduction in JEV titerin host brain which may lead to an extended lifespan orrecovery of infected patient Further diverse mimotopes ofactive virus antigens that can mimic the JEV neutralizingantigen activity can be generated Similarly VLPs and fusionproteins are also used for generation of potential vaccinesFurthermore so many JEV infectious mutant clones canbe generated by insertion of short introns or cloning intoartificial chromosomal systems [191] However for bettertreatment conserved sequence of all different types of struc-tural proteins of JE virus that might challenge acute viralinfections with multiple of overlapping clinical symptomsshould be used for making multivalent vaccines Furtheroccurrence of climate induced genotypic variations or muta-tions and other molecular changes occurred in flavivirusesmust be identified inexcusably added and considered forgenerating new potential vaccines Furthermore therapeuticcould assign a significant effect on virus antigenic sitesand more competitively bind to it However therapeuticbiomarkers can explore the anti-viral potential of vaccinespost vaccination effects seroconversion rate and suppressive


effects of drugs on neurovirulence However for successfulcontrol of JE clinically reliable tests are to be used for properdiagnosis of JE virus in clinical samples with strong pro-phylactic and therapeutic measures Recently whole genomemicroarray research tried tomake neurons tomake their owndefense against Japanese encephalitis viral infection Thesecould work as future regenerative therapeutic markers for JEdisease

12 Conclusion

Unfortunately due to lack of potential biomarkers for JEVdetection and unavailability of timely treatment very highmortality is occurring almost every year in Southeast AsiaBecause of shorter incubation period highmultiplication andinfection rate slow diagnosis and unavailability of timelytreatment high mortality is seen in different parts of Indiaand its neighboring countries [192 193] Further due todemographic and cultural reasons JE is regularly spreadingin non-endemic areas Recently indigenous transmission ofJEV is also observed in urban areas [194] It is fact that inrural areas no JE diagnostic facilities are available to confirmthe disease and in most of the cases due to lack of strongconfirmatory biomarkers patients die with out having anytherapeutic treatment It has lead to an unexpected increasein the morbidity and mortality rates in rural pockets ofIndia Hence there is an utmost need to have strong clinicalbiomarkers to decide the JE disease very fast at an earlierstage Further potential biomarkers are essentially requiredfor deciding cause of disease vaccination and monitoringthe efficacy of therapies Thus by adopting rapid and properdiagnostic tests one can improve the case detection rateclinical index of suspects difference between affected andnon-affected people

Further genotype based neurovirulence antigenicitypathogenesis and mortality must be re-investigated by usingnew candidate markers which can assist in exploration ofpathological mechanisms followed by virus during invasionpersistence and clearance More specifically in absence ofpotential biomarkers many facts regarding JE virus neu-ropathogenesis and other disease related effects are eitherundisclosed or incomplete Further for elimination of infec-tious viruses like JE antigenic modifications are to berequired to generate more appropriate and highly successfulvaccines It will necessitate antigenic profiling of all JE virusesand associating strains to explore the vary reasons of severityof epidemics However standardization of biomarkers willhelp in identification of new JEV mutant strains exist inendemic and JEV prone areas It will also help in riskassessment immunization and postvaccination success andimmune effects in animals and human population to designnew safe vaccines A rapid test that delivers a quick resultshould be followed It will make possible for the physicianto discuss with the patient how to proceed and if necessaryto start treatment immediately after the test In additionstrong confirmatory biomarkers will enable physicians todevelop individualized treatment plans that will help in earlyprimary care of JE patients Conclusively with aid of a perfect

biomarker clinicians will find a clear solution for progressionand type of treatment required for JE patients Naturally thedetection method for a biomarker must be accurate and aseasy to be carried out as possible The results from differentlaboratories may not differ significantly from each other andthe biomarkermust naturally have proven its effectiveness forthe diagnosis prognosis and risk assessment of the affecteddiseases in independent studies A biomarker for clinicaluse needs good sensitivity and specificity It should put apositive predictive value rather than negative for behavioralcare of patient Finally all JE viruses related effects must becorrelated with clinical studies biomarker information andautopsy outcomes for knowing the last stage of severity ofdisease and failure of medicare

Further studies on core candidate markers for JE dis-ease will characterize some more specific unknown factsabout pathological mechanisms of marker regulation andexpression No doubt these will be more differentiated andcomplex than current mechanisms These will provide aclear insight into various levels of JE virus pathogenesisand multidimensional diagnosis based on upgraded tech-nological methods There is another possibility that neuro-viral infections may invite some coinfections and that maycause clinical complications Therefore it is possible that aprotozoan or a bacterial infection will also infect the patientsduring the progression of JE disease However in a stateof disease outbreak by drug resistant microbe and antibodyresistant JE virus strain highly specific biomarkers would berequired to validate the cause of infection However adventof new JEVmarkers could benefit clinician to findmore exactand authentic reasons of risk progression and itsmanagementby applying various therapeutic measures against JE diseaseMoreover disease-related biomarkers should provide anindication about cause of disease probable effect of virusand therapeutic treatment on patient More specifically bothrisk indicator and diagnostic biomarkers will make a balancebetween intensity of infection and possible dose required forthe treatment Furthermore no doubt predictive biomarkerswill provide a real assessment of most likely response of virusand host to a particular treatment type while prognosticmarkers may clear the progression of disease with or withouttreatment Similarly therapeutic biomarkers may correlatedrug-related and vaccine-related effects in patients Thesewill find exact drug response and rate of improvement andmay limit the drug generated toxicadverse effects due toprescription of over or low dose of any therapeutic agent inpatients

Therefore collaborative research efforts are required bothfrom industrial research institutions and from virology peergroups to develop some new innovative biomarkers forfast detection of JE virus in clinical samples There mustbe a better understanding among clinicians molecular andcellular neurobiologists immunologists for starting newresearch initiatives to make landmark innovations in the fieldof biomarker researches Further regenerative biomarkerswould be developed for wound healing of virus injuredneurons in CNS diseases This is only possible by makingfine coordination between interdisciplinary research fieldsmainly cell biologymolecular biology immunology virology


and biomedical sciences Further various socioeconomic andmedico-economic acceptable models of must be designedand used to work out JE risk stratification in endemicpopulation For streamlining valuable insights innovativeresearches to maximize subject resources data acquisitionand multifaceted analysis should be of high priority Nodoubt biomarker studies could explore future perspectives ofclinical diagnosis of JEV infected acute encephalitis patientsand give rise new constraints for successful JE control infuture Hence biomarkers that could help in early diagnosisdisease prevention drug target identification drug responseare to be needed for management of JE disease It willprovide a better safe guard to pediatric groups in providingstrong preventive and therapeuticmeasures For this purposeJEV diagnostic laboratories must be established in endemicareas for timely diagnosis of JE virus to avoid extra delayin treatment It will certainly cut down mortality rate ininfants Further time bound consistent powerful surveillanceof JEV in endemic areas will provide a real time data on itsinfectivity morbidity mortality and clinical care of patientsIn addition more stress must be given on interventionslike mosquito control and avoidance of human exposure tomosquitoes and reservoir hosts can make successful controlof JE disease Therefore strong prophylactic and therapeuticmeasures are required for successful control of JE diseasein endemic areas More specifically biomarker studies maylead to some unexpected results unknown multidimensionalwhich can provide a clear insight on pathophysiology of JEvirus disease in the future Hence strong recommendationsare being made to improve biomarkers used in clinicalpractice and more sophisticated new emerging biomarkersare to be generated and included in clinical diagnosis of JEVinfected acute encephalitis patients and will also give rise tonew constraints for successful JE control

Abbreviations

NS Nonstructural proteinsCSF Cerebrospinal fluidVSV Vesicular stomatitis virusJEV Japanese encephalitis virusTCR T-cell receptorCTL Cytotoxic LymphocytesNCPs Neural Progenitor CellsCNS Central nervous system

Conflict of Interests

The author has no conflict of interests The author alone isresponsible for the content and writing of the paper

References

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[2] P Gunakasem C Chantrasri P Simasathien S ChaiyanunS Jatanasen and A Pariyanonth ldquoSurveillance of Japaneseencephalitis cases in Thailandrdquo The Southeast Asian Journal of

Tropical Medicine and Public Health vol 12 no 3 pp 333ndash3371981

[3] M J Cardosa B H Choo and I Zuraini ldquoA serological studyof Japanese encephalitis virus infections in northern PeninsularMalaysiardquoThe Southeast Asian Journal of Tropical Medicine andPublic Health vol 22 no 3 pp 341ndash346 1991

[4] A Desai V Ravi A Chandramuki and M Gourie-DevildquoDetection of neutralizing antibodies to Japanese encephalitisvirus in the cerebrospinal fluid using a rapid microneutral-ization testrdquo Serodiagnosis and Immunotherapy in InfectiousDisease vol 6 no 3 pp 130ndash134 1994

[5] A Shrivastva N Tripathi M Parida P Dash A Jana andP Lakshmana Rao ldquoComparison of a dipstick enzyme-linkedimmunosorbent assay with commercial assays for detection ofJapanese encephalitis virus-specific IgM antibodiesrdquo Journal ofPostgraduate Medicine vol 54 no 3 pp 181ndash185 2008

[6] N Litzba C S Klade S Lederer and M Niedrig ldquoEvaluationof serological diagnostic test systems assessing the immuneresponse to Japanese encephalitis vaccinationrdquo PLoS NeglectedTropical Diseases vol 4 no 11 article e883 2010

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[8] T Solomon L T T Thao N M Dung et al ldquoRapid diagnosisof Japanese encephalitis by using an immunoglobulin M dotenzyme immunoassayrdquo Journal of Clinical Microbiology vol 36no 7 pp 2030ndash2034 1998

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[10] W Zhou V Pool J K Iskander et al ldquoSurveillance for safetyafter immunization vaccine Adverse Event Reporting Sys-tem (VAERS)mdashUnited States 1991ndash2001rdquoMMWR SurveillanceSummaries vol 2452 no 1 pp 1ndash24 2003

[11] S-B Zhang P Li and X-Z Liu ldquoAnalysis on neutralizationantibody titer of Japanese B encephalitis virus in healthypopulation in Shaanxi provincerdquo Zhongguo Yi Miao He MianYi vol 16 no 3 pp 251ndash257 2010

[12] M Saini and S Vrati ldquoA Japanese encephalitis virus peptidepresent on Johnson grass mosaic virus-like particles inducesvirus-neutralizing antibodies and protects mice against lethalchallengerdquo Journal of Virology vol 77 no 6 pp 3487ndash34942003

[13] K Wang and V Deubel ldquoMice with different susceptibility toJapanese encephalitis virus infection show selective neutralizingantibody response and myeloid cell infectivityrdquo PLoS ONE vol6 no 9 Article ID e24744 2011

[14] R Kaur G Sachdeva and S Vrati ldquoPlasmid DNA immuniza-tion against Japanese encephalitis virus immunogenicity ofmembrane-anchored and secretory envelope proteinrdquo Journalof Infectious Diseases vol 185 no 1 pp 1ndash12 2002

[15] E KonishiM Yamaoka K-S Khin-Sane-Win I Kurane and PW Mason ldquoInduction of protective immunity against japaneseencephalitis in mice by immunization with a plasmid encodingJapanese encephalitis virus premembrane and envelope genesrdquoJournal of Virology vol 72 no 6 pp 4925ndash4930 1998

[16] E Konishi N Ajiro C Nukuzuma P WMason and I KuraneldquoComparison of protective efficacies of plasmidDNAs encodingJapanese encephalitis virus proteins that induce neutralizingantibody or cytotoxic T lymphocytes in micerdquo Vaccine vol 21no 25-26 pp 3675ndash3683 2003


[17] A A Marucci and T C Fuller ldquoQuantitative micro-com-plement fixation testrdquo Applied Microbiology vol 21 no 2 pp260ndash264 1971

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[19] T Takegami H Miyamoto H Nakamura and K YasuildquoDifferences in biological activity of the V3 envelope protein oftwo Japanese encephalitis virus strainsrdquoActa Virologica vol 26no 5 pp 321ndash327 1982

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[24] J T Roehrig J H Mathews and D W Trent ldquoIdentification ofepitopes on the E glycoprotein of Saint Louis encephalitis virususing monoclonal antibodiesrdquo Virology vol 128 no 1 pp 118ndash126 1983

[25] J J Schlesinger and M W Brandriss ldquo17D yellow fever virusinfection of P388D1 cells mediated by monoclonal antibodiesproperties of the macrophage Fc receptorrdquo Journal of GeneralVirology vol 64 no 6 pp 1255ndash1262 1983

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[32] S K Anuradha Y A Surekha S Narayan et al ldquoJapaneseencephalitis virus common cause of viral encephalitis in paedi-atric age group in Bellary Karnataka Indiardquo Journal of Clinicaland Diagnostic Research vol 5 no 3 pp 480ndash482 2011

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[54] I Kurane B L Innis A Nisalak et al ldquoHuman T cell responseto dengue virus antigens Proliferative responses and interferongamma productionrdquo Journal of Clinical Investigation vol 83 no2 pp 506ndash513 1989

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[65] R Feuer I Mena R R Pagarigan S Harkins D E Hassettand J L Whitton ldquoCoxsackievirus B3 and the neonatal CNSthe roles of stem cells developing neurons and apoptosis ininfection viral dissemination and diseaserdquo American Journalof Pathology vol 163 no 4 pp 1379ndash1393 2003

[66] R Feuer R R Pagarigan S Harkins F Liu I P Hunziker andJ L Whitton ldquoCoxsackievirus targets proliferating neuronalprogenitor cells in the neonatal CNSrdquo Journal of Neurosciencevol 25 no 9 pp 2434ndash2444 2005

[67] I Kosugi Y Shinmura H Kawasaki et al ldquoCytomegalovirusinfection of the central nervous system stem cells from mouseembryo a model for developmental brain disorders induced bycytomegalovirusrdquo Laboratory Investigation vol 80 no 9 pp1373ndash1383 2000

[68] S Das and A Basu ldquoInflammation a new candidate in modu-lating adult neurogenesisrdquo Journal of Neuroscience Research vol86 no 6 pp 1199ndash1208 2008

[69] S Das and A Basu ldquoJapanese encephalitis virus infects neuralprogenitor cells and decreases their proliferationrdquo Journal ofNeurochemistry vol 106 no 4 pp 1624ndash1636 2008

[70] A Ghoshal S Das S Ghosh et al ldquoProinflammatorymediatorsreleased by activated microglia induces neuronal death inJapanese encephalitisrdquo GLIA vol 55 no 5 pp 483ndash496 2007

[71] V Swarup S Das S Ghosh and A Basu ldquoTumor necrosisfactor receptor-1-induced neuronal death by TRADD con-tributes to the pathogenesis of Japanese encephalitisrdquo Journalof Neurochemistry vol 103 no 2 pp 771ndash783 2007

[72] J J Wang C L Liao C I Yang Y L Lin C T Chiou and LK Chen ldquoLocalizations of NS3 and E proteins in mouse braininfected with mutant strain of Japanese encephalitis virusrdquoArchives of Virology vol 143 no 12 pp 2353ndash2369 1998

[73] T-C Yang S-L Shiu P-H Chuang et al ldquoJapanese encephali-tis virus NS2B-NS3 protease induces caspase 3 activation andmitochondria-mediated apoptosis in human medulloblastomacellsrdquo Virus Research vol 143 no 1 pp 77ndash85 2009

[74] M KMishra andA Basu ldquoMinocycline neuroprotects reducesmicroglial activation inhibits caspase 3 induction and viralreplication following Japanese encephalitisrdquo Journal of Neuro-chemistry vol 105 no 5 pp 1582ndash1595 2008

[75] X Deng Z Shi S Li et al ldquoCharacterization of nonstructuralprotein 3 of a neurovirulent Japanese encephalitis virus strainisolated from a pigrdquo Virology Journal vol 9 no 8 article 2092011

[76] S Vrati V Agarwal P Malik S A Wani and M SainildquoMolecular characterization of an Indian isolate of Japaneseencephalitis virus that shows an extended lag phase duringgrowthrdquo Journal of General Virology vol 80 no 7 pp 1665ndash16711999

[77] K Yasui ldquoNeuropathogenesis of Japanese encephalitis virusrdquoJournal of NeuroVirology vol 8 no 2 pp 112ndash114 2002

[78] B A Reynolds and S Weiss ldquoClonal and population analysesdemonstrate that an EGF-responsive mammalian embryonicCNS precursor is a stem cellrdquo Developmental Biology vol 175no 1 pp 1ndash13 1996

[79] R J Felling M J Snyder M J Romanko et al ldquoNeuralstemprogenitor cells participate in the regenerative responseto perinatal hypoxiaischemiardquo Journal of Neuroscience vol 26no 16 pp 4359ndash4369 2006

[80] I Schmid S W Cole Y D Korin J A Zack and J V GiorgildquoDetection of cell cycle subcompartments by flow cytometricestimation of DNA-RNA content in combination with dual-color immunofluorescencerdquo Cytometry vol 1 no 39 pp 108ndash116 2000

[81] S J Morrison N M Shah and D J Anderson ldquoRegulatorymechanisms in stem cell biologyrdquo Cell vol 88 no 3 pp 287ndash298 1997

[82] H Sooryanarain V Ayachit and M Gore ldquoActivated CD56(+)lymphocytes (NK+NKT) mediate immunomodulatory andanti-viral effects during Japanese encephalitis virus infection ofdendritic cells in vitrordquo Virology vol 25 no 432 pp 250ndash2602012


[83] A G Aleyas Y W Han J A George et al ldquoMultifront assaulton antigen presentation by Japanese encephalitis virus subvertsCD8+ T cell responsesrdquo Journal of Immunology vol 185 no 3pp 1429ndash1441 2010

[84] A G Aleyas Y W Han A M Patil et al ldquoImpaired cross-presentation of CD8120572+ CD11c+ dendritic cells by Japaneseencephalitis virus in a TLR2MyD88 signal pathway dependentmannerrdquo European Journal of Immunology vol 42 no 10 pp2655ndash2666 2012

[85] Y Z Zhai Y Zhou L Ma and G H Feng ldquoThe dominantroles of ICAM-1-endcoding gene in DNA vaccination againstJapanese encephalitis virus are the activation of dendritic cellsand enhancement of cellular immunityrdquo Cellular Immunologyvol 28 no 1 10 pages 2013

[86] T Thongtan N Wikan P Wintachai et al ldquoCharacterizationof putative Japanese encephalitis virus receptor molecules onmicroglial cellsrdquo Journal of Medical Virology vol 84 no 4 pp615ndash623 2012

[87] C-J Chen Y-C Ou C-Y Chang et al ldquoTNF-120572 and IL-1120573 mediate Japanese encephalitis virus-induced RANTES geneexpression in astrocytesrdquo Neurochemistry International vol 58no 2 pp 234ndash242 2011


[89] C A van de Weg E s de Araujo H J van den Ham et alldquoMicrobial translocation is associated with extensive immuneactivation in dengue virus infected patients with severe diseaserdquoPLOSNeglected Tropical Diseases vol 23 no 7 Article ID e22362013

[90] N Gupta V Lomash and P V L Rao ldquoExpression profile ofJapanese encephalitis virus induced neuroinflammation and itsimplication in disease severityrdquo Journal of Clinical Virology vol49 no 1 pp 4ndash10 2010

[91] Y F Tseng C C Wang S K Liao C K Chuang and W JChen ldquoAutomimmunity related demyelination in infection byJapanese encephalitis virusrdquo Journal of Biomedical Science vol28 no 18 p 20 2011

[92] M K Mishra K Dutta S K Saheb and A Basu ldquoUnder-standing the molecular mechanism of blood-brain barrierdamage in an experimental model of Japanese encephalitiscorrelation with minocycline administration as a therapeuticagentrdquo Neurochemistry International vol 55 no 8 pp 717ndash7232009

[93] N Tanuma ldquoCSF biomarekrs in children with acute enceph-alopathy syndromerdquo Nihon Rinsho vol 70 no 5 pp 887ndash8922012

[94] S P Argade and K Banerjee ldquoPlasma lactic dehydrogenase inmice infected with Japanese encephalitis andWest Nile virusesrdquoIndian Journal of Medical Research A vol 91 pp 307ndash314 1990

[95] C Y Lai YC qui C Y Chang H C Pan et al ldquoEndothelialJapanese encephalitis virus infection enhances migration andadhesion of leukocytes to brain microvascular endothelia viaMEK-dependent expression of ICAM1 and the CINC andRANTES chemokinesrdquo Journal of Neurochemistry vol 123 no2 pp 250ndash261 2012

[96] S V Bardina and J K Lim ldquoThe role of chemokines inthe pathogenesis of neurotropic flavivirusesrdquo ImmunologicResearch vol 54 no 1ndash3 pp 1ndash12 2012

[97] T-C Yang C-C Lai S-L Shiu et al ldquoJapanese encephalitisvirus down-regulates thioredoxin and induces ROS-mediated

ASK1-ERKp38 MAPK activation in human promonocytecellsrdquoMicrobes and Infection vol 12 no 8-9 pp 643ndash651 2010

[98] T Valyi-Nagy and T S Dermody ldquoRole of oxidative damagein the pathogenesis of viral infections of the nervous systemrdquoHistology and Histopathology vol 20 no 3 pp 957ndash967 2005

[99] R Srivastava J KalitaMYKhan andUKMisra ldquoFree radicalgeneration by neurons in rat model of Japanese encephalitisrdquoNeurochemical Research vol 34 no 12 pp 2141ndash2146 2009

[100] S Cao Y Li J Ye et al ldquoJapanese encephalitis virus wild straininfection suppresses dendritic cells maturation and functionand causes the expansion of regulatory T cellsrdquoVirology Journalvol 26 no 8 article 39 2011

[101] M Sinniah ldquoA review of Japanese-B virus encephalitis inMalaysiardquoThe Southeast Asian Journal of Tropical Medicine andPublic Health vol 20 no 4 pp 581ndash585 1989

[102] B Kang J Oh C Lee et al ldquoEvaluation of a rapid immunodi-agnostic test kit for rabies virusrdquo Journal of Virological Methodsvol 145 no 1 pp 30ndash36 2007

[103] E A Gould and A Buckley ldquoAntibody-dependent enhance-ment of yellow fever and Japanese encephalitis virus neuroviru-lencerdquo Journal of General Virology vol 70 no 6 pp 1605ndash16081989

[104] E A Gould A Buckley B K Groeger P A Cane andM Doenhoff ldquoImmune enhancement of yellow fever virusneurovirulence for mice Studies of mechanisms involvedrdquoJournal of General Virology vol 68 no 12 pp 3105ndash3112 1987

[105] K D Yang W-T Yeh R-F Chen et al ldquoA model to studyneurotropism and persistency of Japanese encephalitis virusinfection in human neuroblastoma cells and leukocytesrdquo Jour-nal of General Virology vol 85 no 3 pp 635ndash642 2004

[106] Y Kumar Liang Z Bo J C Rajapakse E E Ooi and SR Tannenbaum ldquoSerum proteome and cytokine analysis in alongitudinal cohort of adults with primary dengue infectionreveals predictive markers of DHFrdquo PLOS Neglected TropicalDiseases vol 6 no 11 Article ID e1887 2012

[107] T P Endy and A Nisalak ldquoJapanese encephalitis virus ecol-ogy and epidemiologyrdquo Current Topics in Microbiology andImmunology vol 267 pp 11ndash48 2002

[108] M Gromeier E Wimmer and A E Gorbalenya ldquoGeneticspathogenesis and evolution of picornavirusesrdquo in Origin andEvolution of Viruses E Domingo R G Webster and J JHolland Eds pp 287ndash343 Academic Press London UK 1999

[109] T J Chambers C S Hahn R Galler and C M RiceldquoFlavivirus genome organization expression and replicationrdquoAnnual Review of Microbiology vol 44 pp 649ndash688 1990

[110] C Lin S M Amberg T J Chambers and C M RiceldquoCleavage at a novel site in the NS4A region by the yellow fevervirus NS2B-3 proteinase is a prerequisite for processing at thedownstream 4A4B signalase siterdquo Journal of Virology vol 67no 4 pp 2327ndash2335 1993

[111] CM Rice EM Lenches and S R Eddy ldquoNucleotide sequenceof yellow fever virus implications for flavivirus gene expressionand evolutionrdquo Science vol 229 no 4715 pp 726ndash733 1985

[112] C M Rice E G Strauss and J H Strauss ldquoStructure ofthe flavivirus genomerdquo in The Togaviridae and Flaviviridae SSchlesinger and M J Schlesinger Eds pp 279ndash326 PlenumPress New York NY USA 1986

[113] H Sumiyoshi C Mori I Fuke et al ldquoComplete nucleotidesequence of the Japanese encephalitis virus genome RNArdquoVirology vol 161 no 2 pp 497ndash510 1987


[114] T Yamashita H Unno Y Mori et al ldquoCrystal structureof the catalytic domain of Japanese encephalitis virus NS3helicasenucleoside triphosphatase at a resolution of 18 ArdquoVirology vol 373 no 2 pp 426ndash436 2008

[115] D A Muller and P R Young ldquoThe flavivirus NS1 proteinmolecular and structural biology immunology role in patho-genesis and application as a diagnostic biomarkerrdquo AntiviralResearch vol 98 no 2 pp 192ndash208 2013

[116] Y-Q Deng J-X Dai G-H Ji et al ldquoA broadly flaviviruscross-neutralizingmonoclonal antibody that recognizes a novelepitope within the fusion loop of E proteinrdquo PLoS ONE vol 6no 1 Article ID e16059 2011


[118] E A Gould A Buckley A D T Barrett and N CammackldquoNeutralizing (54K) and non-neutralizing (54K and 48K)monoclonal antibodies against structural and non-structuralyellow fever virus proteins confer immunity in micerdquo Journalof General Virology vol 67 no 3 pp 591ndash595 1986

[119] M W Brandriss J J Schlesinger E E Walsh and M BrisellildquoLethal 17D yellow fever encephalitis in mice I Passive protec-tion by monoclonal antibodies to the envelope proteins of 17Dyellow fever and dengue 2 virusesrdquo Journal of General Virologyvol 67 no 2 pp 229ndash234 1986

[120] D A Muller S R Corrie J Coffey P R Young and MA Kendall ldquoSurface modified microprojection arrays for theselective extraction of the dengue virus NS1 protein as a markerfor diseaserdquo Analytical Chemistry vol 84 no 7 pp 3262ndash32682012

[121] D A Muller F D Frentiu A Rojas L A Moreira S L OrsquoNeilland P R Young ldquoA portable approach for the surveillanceof dengue virus-infected mosquitoesrdquo Journal of VirologicalMethods vol 183 no 1 pp 90ndash93 2012

[122] J J SchlesingerMW Brandriss and E EWalsh ldquoProtection ofmice against dengue 2 virus encephalitis by immunization withthe dengue 2 virus non-structural glycoprotein NS1rdquo Journal ofGeneral Virology vol 68 no 3 pp 853ndash857 1987

[123] E A Henchal L S Henchal and B K ThaisomboonsukldquoTopological mapping of unique epitopes on the dengue-2 virusNS1 protein using monoclonal antibodiesrdquo Journal of GeneralVirology vol 68 no 3 pp 845ndash851 1987

[124] B Falgout M Bray J J Schlesinger and C-J Lai ldquoImmu-nization of mice with recombinant vaccinia virus expressingauthentic dengue virus nonstructural protein NS1 protectsagainst lethal dengue virus encephalitisrdquo Journal of Virology vol64 no 9 pp 4356ndash4363 1990

[125] P AvirutnanN Punyadee S Noisakran et al ldquoVascular leakagein severe dengue virus infections a potential role for thenonstructural viral protein NS1 and complementrdquo Journal ofInfectious Diseases vol 193 no 8 pp 1078ndash1088 2006

[126] D-S Sun C-C King H-S Huang et al ldquoAntiplatelet autoan-tibodies elicited by dengue virus non-structural protein 1cause thrombocytopenia and mortality in micerdquo Journal ofThrombosis and Haemostasis vol 5 no 11 pp 2291ndash2299 2007

[127] M Bray B Zhao L Markoff K H Eckels R M Chanock andC-J Lai ldquoMice immunized with recombinant vaccinia virusexpressing dengue 4 virus structural proteins with or withoutnonstructural protein NS1 are protected against fatal denguevirus encephalitisrdquo Journal of Virology vol 63 no 6 pp 2853ndash2856 1989

[128] Y-L Lin L-K Chen C-L Liao et al ldquoProtective immunity inmice virus nonstructural protein NS1 elicits DNA immuniza-tion with Japanese encephalitisrdquo Journal of Virology vol 72 no1 pp 191ndash200 1998

[129] E Konishi S Pincus B A L Fonseca R E Shope E Paolettiand P W Mason ldquoComparison of protective immunity elicitedby recombinant vaccinia viruses that synthesize E or NS1 ofJapanese encephalitis virusrdquo Virology vol 185 no 1 pp 401ndash410 1991

[130] J J Schlesinger M W Brandriss and E E Walsh ldquoProtectionagainst 17D yellow fever encephalitis in mice by passive transferof monoclonal antibodies to the nonstructural glycoproteingp48 and by active immunization with gp48rdquo Journal ofImmunology vol 135 no 4 pp 2805ndash2809 1985

[131] J J Schlesinger MW Brandriss C B Cropp and T P MonathldquoProtection against yellow fever in monkeys by immunizationwith yellow fever virus nonstructural protein NS1rdquo Journal ofVirology vol 60 no 3 pp 1153ndash1155 1986

[132] H Kawano V Rostapshov L Rosen and C-J Lai ldquoGeneticdeterminants of dengue type 4 virus neurovirulence for micerdquoJournal of Virology vol 67 no 11 pp 6567ndash6575 1993

[133] J M Mackenzie M K Jones and P R Young ldquoImmunolo-calization of the Dengue virus nonstructural glycoprotein NS1suggests a role in viral RNA replicationrdquo Virology vol 220 no1 pp 232ndash240 1996

[134] E G Westaway J M Mackenzie M T Kenney M K Jonesand A A Khromykh ldquoUltrastructure of Kunjin virus-infectedcells colocalization ofNS1 andNS3with double-stranded RNAand of NS2B with NS3 in virus-inducedmembrane structuresrdquoJournal of Virology vol 71 no 9 pp 6650ndash6661 1997

[135] G Winkler V B Randolph G R Cleaves T E Ryan andV Stollar ldquoEvidence that the mature form of the flavivirusnonstructural protein NS1 is a dimerrdquo Virology vol 162 no 1pp 187ndash196 1988

[136] P W Mason ldquoMaturation of Japanese encephalitis virus glyco-proteins produced by infected mammalian and mosquito cellsrdquoVirology vol 169 no 2 pp 354ndash364 1989

[137] G W Smith and P J Wright ldquoSynthesis of proteins andglycoproteins in dengue type 2 virus-infected vero and Aedesalbopictus cellsrdquo Journal of General Virology vol 66 no 3 pp559ndash571 1985

[138] E G Westaway and M R Goodman ldquoVariation in distribu-tion of the three flavivirus-specified glycoproteins detected byimmunofluorescence in infected vero cellsrdquoArchives of Virologyvol 94 no 3-4 pp 215ndash228 1987

[139] I Gutsche F Coulibaly J E Voss et al ldquoSecreted dengue virusnonstructural protein NS1 is an atypical barrel-shaped high-density lipoproteinrdquo Proceedings of the National Academy ofSciences of theUnited States of America vol 108 no 19 pp 8003ndash8008 2011

[140] L-K Chen C-L Liao C-G Lin et al ldquoPersistence of Japaneseencephalitis virus is associated with abnormal expression of thenonstructural protein NS1 in host cellsrdquo Virology vol 217 no 1pp 220ndash229 1996

[141] J-J Hung M-T Hsieh M-J Young C-L Kao C-C Kingand W Chang ldquoAn external loop region of domain III ofdengue virus type 2 envelope protein is involved in serotype-specific binding to mosquito but not mammalian cellsrdquo Journalof Virology vol 78 no 1 pp 378ndash388 2004

[142] AGoto K YoshiiMObara et al ldquoRole of theN-linked glycansof the prM and E envelope proteins in tick-borne encephalitis


virus particle secretionrdquo Vaccine vol 23 no 23 pp 3043ndash30522005

[143] H-YWei L-F Jiang D-Y Fang andH-Y Guo ldquoDengue virustype 2 infects human endothelial cells through binding of theviral envelope glycoprotein to cell surface polypeptidesrdquo Journalof General Virology vol 84 no 11 pp 3095ndash3098 2003

[144] M K Jeong I Y Sang H S Byung et al ldquoA single-linkedglycosilation site in the Japanese encephalitis virus prM proteinis critical for cell type-specific prM protein biogenesis virusparticle release and pathogenicity in micerdquo Journal of Virologyvol 82 no 16 pp 7846ndash7862

[145] M Gromeier B Bossert M Arita A Nomoto and EWimmerldquoDual stem loopswithin the poliovirus internal ribosomal entrysite control neurovirulencerdquo Journal of Virology vol 73 no 2pp 958ndash964 1999


[147] M Bray and C-J Lai ldquoConstruction of intertypic chimericdengue viruses by substitution of structural protein genesrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 88 no 22 pp 10342ndash10346 1991

[148] L J Johnston G M Halliday and N J King ldquoLangerhans cellsare targets of dengue virus infectionrdquo Nature Medicine vol 6pp 816ndash820 2000

[149] Y Zhang P Chen R Cao and J Gu ldquoMutation of putativeN-linked glycosylation sites in japanese encephalitis virus pre-membrane and envelope proteins enhances humoral immunityin BALBC mice after DNA vaccinationrdquo Virology Journal vol8 article 138 2011

[150] M Zhang B Gaschen W Blay et al ldquoTracking global patternsof N-linked glycosylation site variation in highly variable viralglycoproteins HIV SIV and HCV envelopes and influenzahemagglutininrdquo Glycobiology vol 14 no 12 pp 1229ndash12462004

[151] A Goffard N Callens B Bartosch et al ldquoRole of N-linkedglycans in the functions of hepatitis C virus envelope glycopro-teinsrdquo Journal of Virology vol 79 no 13 pp 8400ndash8409 2005

[152] R W Schlesinger ldquoVirus-host interactions in natural andexperimental infections with alphaviruses and flavivirusesrdquo inThe Togaviruses R W Schlesinger Ed pp 83ndash104 AcademicPress New York NY USA 1980

[153] V I Agol S G Drozdov and V P Grachev ldquoRecombinantsbetween attenuated and virulent strains of poliovirus type 1derivation and characterization of recombinants with centrallylocated crossover pointsrdquo Virology vol 143 no 2 pp 467ndash4771985

[154] T Omata M Kohara S Kuge et al ldquoGenetic analysis of theattenuation phenotype of poliovirus type 1rdquo Journal of Virologyvol 58 no 2 pp 348ndash358 1986

[155] M Tardy-Panit B Blondel A Martin F Tekaia F Horaudand F Delpeyroux ldquoA mutation in the RNA polymerase ofpoliovirus type 1 contributes to attenuation in micerdquo Journal ofVirology vol 67 no 8 pp 4630ndash4638 1993

[156] P Thomas J A Monath A Juan et al ldquoSingle mutation in theflavivirus envelope protein hinge region increases neuroviru-lence for mice and monkeys but decreases viscerotropism formonkeys relevance to development and safety testing of liveattenuated vaccinesrdquo Journal of Virology vol 76 no 4 pp 1932ndash1943 2002

[157] M Ahmed T R Marino S Puckett N D Kock and D SLyles ldquoImmune response in the absence of neurovirulence inmice infected withMproteinmutant vesicular stomatitis virusrdquoJournal of Virology vol 82 no 18 pp 9273ndash9277 2008

[158] J J Schlesinger S Chapman A Nestorowicz C M Rice TE Ginocchio and T J Chambers ldquoReplication of yellow fevervirus in the mouse central nervous system comparison of neu-roadapted and non-neuroadapted virus and partial sequenceanalysis of the neuroadapted strainrdquo Journal of General Virologyvol 77 no 6 pp 1277ndash1285 1996



[161] Y Yang J Ye X Yang R Jiang H Chen and S Cao ldquoJapaneseencephalitis virus infection induces changes ofmRNAprofile ofmouse spleen and brainrdquo Virology Journal vol 24 no 8 p 802011

[162] G C Sahoo M R Dikhit and P Das ldquoFunctional assignmentto JEV proteins using SVMrdquoBioinformation vol 3 no 1 pp 1ndash72008


[164] E Konishi M Yamaoka I Kurane and P W Mason ldquoJapaneseencephalitis DNA vaccine candidates expressing premembraneand envelope genes induce virus-specific memory B cells andlong-lasting antibodies in swinerdquo Virology vol 268 no 1 pp49ndash55 2000

[165] S-C Wu and C-W Lin ldquoNeutralizing peptide ligands selectedfrom phage-displayed libraries mimic the conformational epi-tope on domain III of the Japanese encephalitis virus envelopeproteinrdquo Virus Research vol 76 no 1 pp 59ndash69 2001

[166] M Liu H Chen F Luo et al ldquoDeletion of N-glycosylationsites of hepatitis C virus envelope protein E1 enhances specificcellular and humoral immune responsesrdquo Vaccine vol 25 no36 pp 6572ndash6580 2007

[167] C-W Lin C-W Cheng T-C Yang et al ldquoInterferon antagonistfunction of Japanese encephalitis virusNS4A and its interactionwithDEAD-box RNAhelicase DDX42rdquoVirus Research vol 137no 1 pp 49ndash55 2008

[168] T P Monath J Arroyo I Levenbook et al ldquoSingle mutation inthe flavivirus envelope protein hinge region increases neurovir-ulence for mice and monkeys but decreases viscerotropism formonkeys relevance to development and safety testing of liveattenuated vaccinesrdquo Journal of Virology vol 76 no 4 pp 1932ndash1943 2002

[169] J Arroyo F Guirakhoo S Fenner Z-X Zhang T P Monathand T J Chambers ldquoMolecular basis for attenuation of neu-rovirulence of a yellow fever virusJapanese encephalitis viruschimera vaccine (ChimeriVax-JE)rdquo Journal of Virology vol 75no 2 pp 934ndash942 2001

[170] D-K Yang C-H Kweon B-H Kim et al ldquoTaqman reversetranscription polymerase chain reaction for the detection of


Japanese encephalitis virusrdquo Journal of Veterinary Science vol5 no 4 pp 345ndash351 2004

[171] S Paranjpe AWalimbe and K Banerjee ldquoStatistical analysis ofthe envelope gene and the prM region of Japanese encephalitisvirus evidence suggestive of positive selectionrdquo Journal ofGenetics vol 76 no 2 pp 117ndash131 1997

[172] MBAppaiahgari and SVrati ldquoDNAzyme-mediated inhibitionof Japanese encephalitis virus replication in mouse brainrdquoMolecular Therapy vol 15 no 9 pp 1593ndash1599 2007

[173] Y Yang J Ye X Yang R Jiang H Chen and S Cao ldquoJapaneseencephalitis virus infection induces changes ofmRNAprofile ofmouse spleen and brainrdquo Virol J vol 24 no 8 p 80 2011

[174] Y Zhang Y Jia R Zheng et al ldquoPlasma microRNA-122 asa biomarker for viral- alcohol- and chemical-related hepaticdiseasesrdquoClinical Chemistry vol 56 no 12 pp 1830ndash1838 2010

[175] R L Skalsky and B R Cullen ldquoViruses microRNAs and hostinteractionsrdquo Annual Review of Microbiology vol 64 pp 123ndash141 2010

[176] A Turchinovich L Weiz A Langheinz and B BurwinkelldquoCharacterization of extracellular circulating microRNArdquoNucleic Acids Research vol 39 no 16 pp 7223ndash7233 2011

[177] A Gupta P Nagilla H-S Le et al ldquoComparative expressionprofile of miRNA and mRNA In primary peripheral bloodmononuclear cells infected with human immunodeficiencyvirus (HIV-1)rdquo PLoS ONE vol 6 no 7 Article ID e22730 2011

[178] M A Prescott andM K Pastey ldquoIdentification of unique bloodand urine biomarkers in influenza virus and Staphylococcusaureus co-infection a preliminary studyrdquo Biomarker Insightsvol 2010 no 5 pp 145ndash151 2010

[179] V Swarup J Ghosh M KMishra and A Basu ldquoNovel strategyfor treatment of Japanese encephalitis using arctigenin a plantlignanrdquo Journal of Antimicrobial Chemotherapy vol 61 no 3pp 679ndash688 2008

[180] K Dutta D Ghosh and A Basu ldquoCurcumin protects neuronalcells from japanese encephalitis virus-mediated cell death andalso inhibits infective viral particle formation by dysregula-tion of ubiquitin-proteasome systemrdquo Journal of NeuroimmunePharmacology vol 4 no 3 pp 328ndash337 2009

[181] E O Era H H Askling et al ldquoCross protective capacity ofJapanese encephalitis (JE) vaccine against circulating heterol-ogous JE virus genotypesrdquo Clinical Infectious Diseases vol 56no 2 pp 267ndash270 2012

[182] U Holmskov S Thiel and J C Jensenius ldquoCollectins andficolins humoral lectins of the innate immune defenserdquoAnnualReview of Immunology vol 21 pp 547ndash578 2003

[183] SD Rosen ldquoLigands for L-selectin homing inflammation andbeyondrdquo Annual Review of Immunology vol 22 pp 129ndash1562004

[184] M W Turner ldquoThe role of mannose-binding lectin in healthand diseaserdquoMolecular Immunology vol 40 no 7 pp 423ndash4292003

[185] K Yamada T Mano Y Inada et al ldquoChanges of brain edemaafter initiation of mild hypothermia therapy in childrenrdquoNo ToHattatsu vol 43 no 1 pp 10ndash13 2011

[186] J-S Zhang Q-M Zhao S-Q Zuo N Jia and X-F GuoldquoCytokine and chemokine responses to Japanese encephalitislive attenuated vaccine in a human populationrdquo InternationalJournal of Infectious Diseases vol 16 no 4 pp e285ndashe288 2012

[187] T Takegami H Miyamoto H Nakamura and K YasuildquoBiological activities of the structural proteins of Japaneseencephalitis virusrdquo Acta Virologica vol 26 no 5 pp 312ndash3201982


[189] S-I Yun S-Y Kim C M Rice and Y-M Lee ldquoDevelopmentand application of a reverse genetics system for Japaneseencephalitis virusrdquo Journal of Virology vol 77 no 11 pp 6450ndash6465 2003

[190] Y-L Lin Y-L Huang S-H Ma et al ldquoInhibition of Japaneseencephalitis virus infection by nitric oxide antiviral effect ofnitric oxide on RNA virus replicationrdquo Journal of Virology vol71 no 7 pp 5227ndash5235 1997

[191] P Kumar K L Sang P Shankar and N Manjunath ldquoA singlesiRNA suppresses fatal encephalitis induced by two differentflavivirusesrdquo PLoS Medicine vol 3 no 4 pp 505ndash514 2006

[192] R K Upadhyay ldquoJapanese encephalitis virus generated neu-rovirulence antigenicity and host immune responsesrdquo ISRNVirology vol 2013 Article ID 830396 24 pages 2013

[193] R K Upadhyay and S Ahmad ldquoJapanese encephalitis virus itsepidemiology disease and vector control with special referenceto immune surveillance and safety measuresrdquo Journal of Phar-macy Research vol 4 no 8 pp 2490ndash2499 2011

[194] R Kumari K Kumar A Rawat G Singh N K Yadav andL S Chauhan ldquoFirst indigenous transmission of Japaneseencephalitis in urban areas of National Capital Territory ofDelhi Indiardquo Tropical Medicine amp International Health vol 18no 6 pp 743ndash749 2013


in pathogenesis in recent past and future Further vaccineattenuated sequence alterations are significantly increasingwhich are not only changing the etiological features of thevirus but are also inducing high neurovirulence and hostimmune responses and affecting disease transmission in bothendemic and nonendemic population However other thanJE virus endemic strains new vaccine-derived recombinantstrains of JE virus have been evolved which are detected inserum samples of patients from endemic regions Howeveroverall changes occurred in JEV have increased the infectionpathogenesis and mortality rate It has also led to an increasein expansion of severity of infection in nonendemic areasAmong other possible reasons of expanding its area areglobal climates ecological and socioeconomic changes andmutations acquired by local strains of JE virus

Though so many methods of JEV detection are availableto days it is very hard to confirm the JE disease based onseeing visible pathological symptoms in patients However itis very clear that disease history of JE infection virus expo-sure and certain clinical features cannot help to diagnose thisdisease properlyThere is an utmost need to develop potentialbiomarkers which might essentially improve the diagnosisand accelerate the development of new vaccinesMore specif-ically biochemical molecular and immunological tools areconsidered to be more authentic and reliable to detect the JEvirus in different tissues and cells [4] These markers couldfacilitate fast diagnosis of JEV invasion in neuronal cellsand behavioral impairments in patients and help to confirmprogression of disease However a clinical diagnosis based ondetermination of the level of virus IgM antibodies expressionof nonstructural viral proteins and pathological changes inneuronal progenitor cells brain cortex thalamus hippocam-pus and striatum will be more helpful in JE confirmation[5] Similarly measurement of the level of certain markers inCSF such as the level of interleukins complements cytokinesand intracellular virus proteins was also found to be usefulin predicting the JEV generated risks and prevalence ofpathogenesis [6] However definitive diagnosis of JE basedon clinical symptoms serology and biochemical profile ofthe virus is quite investigative [7] but it is very difficult todiagnose on going mutations in newly emerging genotypesof JE virus circulating in the endemic population Furthergenotype-based neurovirulence antigenicity pathogenesisand mortality must reinvestigated by using new candidatemarkers Biomarkers will assist in exploration of pathologicalmechanisms followed by virus during invasion persistenceand clearance Therefore strong molecular and proteinbiomarkers are needed for the confirmation of genotype-based infection of JE virus in patients as well as its therapeuticessentiality accordingly

However biomarkers based on measurement of a highlyspecific biomolecule that may confirm the presence of JEvirus pathogen inside host will clearly display the physiolog-ical state of patient Biomolecules synthesized in response toJE virus attack and cellular invasion will show clear biologicalcharacteristics whose measurement in body fluid cells andtissues will confirm the viral disease [4 5] For exampleformation of C-reactive protein (CRP) in response to JEvirus attack works as a marker for inflammation Further

all affected specific cells molecules or genes gene productsenzymes or hormones complex organ functions charac-teristic changes that occurred in the biological structureand function of Biomolecules could serve as biomarkersTherefore biomarkers that clearly indicate and reflect theseverity and status of viral infection in patents will bemore appropriate More specifically such biomarkers couldcorrelate virus-induced changeswith the risk and progressionof JE disease and susceptibility of virus to a given treatmentSimilarly imaging biomarkers (CT PET and MRI) andnonimaging biomarkers could also explore biophysical prop-erties of virus and bio-molecules with their measurements inbiological samples (eg plasma serum cerebrospinal fluidbrain biopsy)

More specifically nucleic acid-based biomarkers suchas gene mutations or polymorphisms and quantitative geneexpression analysis virus-induced peptides proteins lipidsmetabolites and other small molecules could help in earlydiagnosis of JE However gene-based biomarker will be moreeffective and an acceptable marker and may prove better JEdiagnostic tools for fast clinical assessment of JEV in thefuture Furthermore subset of markers that might discoversecretes of virus invasion and cellular pathogenesis by usinggenomics proteomics technologies or imaging technolo-gies would play major roles in medicinal biology Furtherstrong therapeutic pharmacodynamic (PD) biomarkers willbe required for decision making and drug developmentpharmacological response and dose optimization Howeverto have an early clinical investigation ready highly specificbiomarkers are essentially required to display the JE infectionand physiological state of patient before starting certainmedicare Hence in the present review article newly emerg-ing biomarkers for JE are highlighted with their diagnosticefficacy specifications authentication and working accuracywith an objective to recognize cellular abnormalities occur-ring in the JE infected patientsThese biomarkersmight proveto be significant diagnostic tools for JE confirmation in thefuture

2 Cerebrospinal Fluid Biomarkers

JE virus cultured in the host body cells releases somany com-ponents in the cerebrospinal fluid which serve as biomark-ers Hence both interacting and noninteracting moleculesreleased by the cells in anticipation to virus load and invasionof blood and nerve cells are determined accurately Howeveralot of virus eradicating molecules are released by immunecells and virus toxinsantigens in the CSF are assayed forevaluation of presence of virus intensity and rate of infectionin humanhosts However for detection of JE virus interactingmolecules in cerebrospinal fluid and blood serum samplesare collected from both JE endemic and epidemic areasIn normal population blood serum samples are collectedin the premonsoon season and at the time of outbreakDuring infection season samples are collected twice at atime interval of ten days Patient information should berecorded with 5ndash10 different specifications it should includelocation name age sex vaccinated or not date of onset of








































Table 1 Continued



























3 Serum Biomarkers




4 Plasma Biomarkers





















































































12 Conclusion








Abbreviations




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Table 1 Continued



























3 Serum Biomarkers




4 Plasma Biomarkers





















































































12 Conclusion








Abbreviations




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Table 1 Continued



























3 Serum Biomarkers




4 Plasma Biomarkers





















































































12 Conclusion








Abbreviations




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Table 1 Continued



























3 Serum Biomarkers




4 Plasma Biomarkers





















































































12 Conclusion








Abbreviations




References
















































































































































































































3 Serum Biomarkers




4 Plasma Biomarkers





















































































12 Conclusion








Abbreviations




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12 Conclusion








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12 Conclusion








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12 Conclusion








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12 Conclusion








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12 Conclusion








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12 Conclusion








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Biomarkers in Japanese Encephalitis: A Review

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