DENGUEDIAGNOSISA multi-pronged attack

www.medicine.um.edu.my

Dengue

Dengue, in recent decades has becomeone of the most uncontrolled neglectedinfectious diseases especially in thetropical and sub-tropical regions of theworld. The earliest records on diseasesthat were clinically compatible todengue were from the Chin Dynasty(265 to 420 A.D.) and from NorthernSung Dynasty (992 A.D.). Since 1960s,dengue incidences around the worldhave increased by more than 30-fold,with more than 100 countries in allcontinents being affected. Recently, itwas estimated that approximately 390million dengue infections occurs yearly.It is believed that the societal andecological changes/movement duringthe world wars increased vector-bornediseases, and dengue hyperendemicitybegan in the Southeast Asian regions,therein triggering epidemics of DHF.Dengue viruses can be asymptomaticor inapparent, or may cause a widespectrum of symptoms that may rangefrom classical fever to plasma leakage,hemorrhage, shock and even death.Dengue, despite being around forcenturies, has neither commercialisedvaccines nor anti-dengue drugs. Thisrelatively slow progress in dengue vaccination and drug discovery is mainlybecause (i) the vaccine needs to be ableto protect against all 4 DENV serotypes;(ii) the lack of protective immune correlates; (iii) the absence of reliableanimal models to represent dengueand (iv) the controversial and limitedunderstanding of dengue pathogenesis.

Dengue – Laboratory diagnosis

Dengue diagnosis is not only important forclinical management of patients, but alsofor epidemiological surveillance, outbreakintervention and vaccine development andmonitoring. Due to the absence of pathogno-monic clinical features that can distinguishdengue from other febrile illnesses, laboratoryconfirmation is an essential part of diagnosingdengue. Ideally, a dengue diagnostic test shouldbe rapid, simple, with high sensitivity andspecificity, able to serotype and differentiateprimary and secondary infections. Despite themany efforts to create a single assay that couldconfirm dengue, that goal has not been reached.Nevertheless, dengue diagnostics have come along way, with many researchers around theworld attempting for a more efficient andreliable diagnostic method.

The main hurdle in developing an idealdiagnostic assay for dengue would lie in thecomplicated pathogenesis of dengue and thefact that multiple sequential infections occursin dengue endemic areas. Understanding theclinical conditions of dengue patients is essentialfor appropriate usage of current dengue diagnostics which are mostly serological-based,nucleic acid-based and antigen based. Denguediagnosis is divided into two main phases, theearly phase and the late phase. In the earlyphase, the approach of diagnosis is via viraldetection, viral RT-PCRs, and antigen detection.Meanwhile, the later phase of diagnosis ismainly through serological testing.

We have been actively involved in the development of diagnostic kits. An in-housemethod for the detection of dengue IgM via

Issues that are faced in Dengue: A Disease that requires a multi-prong attack

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IgM capture ELISA, which is simple and canprovide an early diagnosis for dengue infectionand a multiplex dengue viral RNA detectionassays were developed and evaluated. In theevaluations that were carried out, the sensitivityof the assay was found to be advantageous,showing good sensitivity and specificity andthe results obtained were less subjective thanthe HI method.

We have been actively involved in evaluationprojects of dengue diagnostic kits conductedby the WHO and collaborating partnersaround the world as well as with companiesthat are commercialising these kits. Theseevaluations revealed that the NS1 detectionrate is inversely proportional while the IgMdetection rate is directly proportional to thepresence of IgG antibodies. Hence, with suchfindings, this information helps in balancingthe use of NS1 kits which are not entirelysolely dependable as a single assay for thedetection of dengue infection. Combining thisassay with an IgM and/or IgG assay willincrease the sensitivity of detection, especiallyin areas with a higher prevalence of secondarydengue virus infections.

Newer technologies are being applied to fine-tune available diagnostic arrays and alsoto design new assays that fit the ideal testconcept. In collaboration with the Faculty ofEngineering, UM are involved in the applicationof biosensors in detecting dengue. Two papers

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Rathakrishnan A, and Sekaran S D (2013)

have since been published revolving aroundthe use of surface plasmon resonance (SPR)as a medical diagnosis technique with highsensitivity and specificity in detection ofdengue. In this research, a new method basedon SPR is proposed for rapid, 10-minutedetection of the anti-dengue virus in humanserum samples. This novel technique, knownas rapid immunoglobulin M (IgM)-baseddengue diagnostic test, can be utilised quicklyand easily at the point of care. According tothe results, a serum volume of only 1 μl froma dengue patient (as a minimised volume) isrequired to indicate SPR angle variation todetermine the ratio of each dengue serotypein samples with 83-93% sensitivity and 100%specificity. This breakthrough is a steppingstone towards faster and better diagnostic results.

With many tests and assays that have beencarried out, we are still in the search toimprove better diagnostics of dengue infection.Our laboratory thrives to develop and test fornew avenues in making dengue diagnosis morerobust and less cumbersome.

Dengue – The Pathogenesis

These past few decades have seen a surge indengue research – in trying to understand thedisease, the cause and the trigger. However,the findings have been frustrating where manystudies have seen controversial findings andmind-boggling correlations. Our lab has beenpart of the worldwide researchers who haveembarked on finding the cause and correlatesof severe dengue and the occurrence ofasymptomatic dengue cases.

Dengue viruses can evade our immune systemin various manners which may include directimmunosuppression, antigenic variability,passive evasion of the innate immunity andactive inhibition of the system. Another postu-lated dengue pathogenesis is that virulency ofdifferent DENV virus strains cause discrepancyin the levels of dengue severity. Furthermore,the sequence of infection by the 4 DENV mayalso be a risk for manifesting severe symptomsof dengue. Mostly though, dengue has beenbelieved to be an immune-mediated diseasewhereby most severe cases seemed to haveoccurred in secondary infections. A dengue-infected person develops lifelong immunitytowards the first infecting DENV serotype;however, this immunity is only partiallyconferred for heterologous dengue serotypesin subsequent infections. Severe dengue caseshave been linked to secondary infections asevidenced by retrospective clinical studiesand also manifestation of severe dengue inbabies with maternal dengue antibodies. Inthis perspective, the common explanationsfor dengue pathogenesis include (i) dengueantibody-dependent enhancement (ADE); and(ii) cross-reactive cytotoxic T cells whereby insecondary infections T cell activation wouldbe greater and more rapid or via originalantigenic sin to be with lower affinity andavidity towards subsequent heterologousdengue serotypes. Consequently, the atypicalactivation of T cells and reaction of enhancingantibodies can trigger an impromptu/ inappropriate cascade of signaling events,

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Jahanshahi P, Zalnezhad E, Sekaran S D, and Adikan F R M (2014)

creating cytokine storms that have been correlated with severity in dengue infections.Furthermore, various host genetic factors havebeen implied in increased dengue severity suchas vitamin D receptor, TNF, FcγRII, CTLA4,TGFβ1 and the HLA family of genes whileTAP1 and TAP2 have been shown to confersusceptibility to/ protection against dengueseverity. Having a strong virology andimmunology background, we investigatedimmune correlates in dengue severity whichincluded HLA association with dengue susceptibility/protection, T cell responses ofdengue infected patients, cytokine profiling ofdengue patients at different phases of illness.This has resulted in various publications inpeer-reviewed journals including Clinical andVaccine Immunology and PLoS One.

Dengue – Asymptomatic and itsrelatedness to possible protection

Recently, it was estimated that among the 390million cases of dengue annually, about 75% weremild or asymptomatic cases – an implicationthat dengue reservoirs might be larger thanexpected. With the understanding that theasymptomatic dengue cases should not be takenlightly, our lab has investigated the asymptomaticdengue cohort to search for predisposingmarkers that constitute protection or lack ofclinical manifestations in these individuals.Applying microarray and high throughputquantitative PCR to assess the molecular basisof the human genomics, we focused on thegene expressions of immunological responsesin the asymptomatic individuals during dengueinfections. Based on comprehensive bioinfor-matics software Spotfire® DecisionSite used inmicroarray analysis, our study was able to show

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Rathakrishnan A, Wang S M, Hu Y, Khan A M, Ponnampalavanar S, Lum L C S, and Sekaran S D (2012)

each gene’s asymptomatic to symptomatic foldchanges and the comparisons’ statistical signif-icance which was depicted in a volcano plot.The near symmetrical distribution of genes inthe plot provided confidence in reliability ofthe expression values of which was essential inelucidating a significant difference betweenthe dengue patients and the asymptomaticindividuals in terms of their immunologicalgenes’ responses. Our study then proceededto supervised hierarchal clustering whichrevealed a heat map depicting clarity that thegene expression patterns of the asymptomatichousehold members’ were different from thepatients’. We revealed involvement of broaddown-regulation of host defense responses,generally the innate and adaptive immuneresponses and the matrix metalloproteasegenes in asymptomatic individuals againstsymptomatic patients. Although a wide spectrumof genes were down regulated, our study high-lighted genes of TNF α (TNF), IL8, C1S, factorB (CFB), IL2, IL3, IL4, IL5, IL8, IL9, IL10 andIL13, CD80, CD28, and IL18, MMP8, MMP10,MMP12, MMP15, MMP16, and MMP24. Onthe contrary, we also observed selective up-regulation of distinct genes that have beenassociated with protection. Those up regulatedgenes were RANTES (CCL5), MIP-1α(CCL3L1/CCL3L3), MIP-1β (CCL4L1), TGFβ(TGFB), and TIMP1. This study giving thepreliminary information on biomarkers thatshowed differences of the gene expressions of

DENV asymptomatic individuals in comparisonwith the DENV infected patients has since beenaccepted for publication in PLoS One. Ourfindings highlight the potential association ofcertain host genes conferring protectionagainst clinical dengue. DENV infectionamong asymptomatic individuals, as comparedto clinical dengue patients provides myriadinformation with regards to gene expressionof immune correlates. These data are valuableto better explore the mysteries behind thepoorly understood immunopathogenesis ofsubclinical dengue infection.

Dengue – Endothelium dysfunction

More recently, we have endeavored the fieldof endothelium dysfunction of infectiousdiseases. This is mainly spurred by her contin-uous investigations of immunopathogenesisof dengue infection. The hallmark of severedengue is plasma leakage and hemorrhage andthe “organ” crucial for regulating electrolytecontent of intra- and extravascular spaces isthe endothelium layer. The endothelium is alsoan important area of immunity because bloodcoagulation, platelet adhesion, and immunecells interactions occur here.

In vitro, endothelial cells (ECs) have beenshown to be permissive to DENV which was onceagain demonstrated by our team in variousendothelial cells of different origins (including

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Rathakrishnan A, Klekamp B, Wang S M, Komarasamy T V, Natkunam S K, Sathar J, and Sekaran S D (2014)

dermal, hepatic, brain and lungs). Previouslyit has been demonstrated that ECs that areexposed to DENV or dengue antibodies areknown to release various cytokines. We havedemonstrated that exposing ECs to certaincytokines such TNF-α and MCPs-1 seemedto induce vascular permeability, observedvia damage induced on the ECs junctionalproteins. In collaboration with investigators atthe Johns Hopkins University and PUGSOM,we are currently investigating the endothelialdysfunction caused by dengue virus infection.With the purchase of the Electric Cell-substrate Impedance Sensing (ECIS) system,the effect of dengue infections on endothelialcells is being probed real time in a non-labelmethod by monitoring morphological changes,

cell locomotion, and other behaviors directedby the cell’s cytoskeleton. Preliminary resultsindicate that the microvascular ECs examinedwere activated immediately upon dengueinfection at high virus titers. This modulationof the endothelial cells appears to be caused bymodulations of the junctional proteins via theparacellular pathway. Currently, the geneexpression of DENV-infected microvascular ECsas early as the moment of infection is beingscrutinised. In other studies which analysedgene expression of DENV -infected ECs at24-48 hours post infection revealed that thecellular pathway, immune response andinflammation genes were upregulated, whereasthe cytoskeletal and membrane structuregenes were downregulated

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Yeo A S, Azhar N A, Yeow W, Talbot Jr C C, Khan M A, Shankar E M, and Sekaran S D (2014)

In addition to direct virus effect on theendothelial cells, viral infection can alsocause oxidative stress which can also lead toendothelial damage. In dengue, serum NO inDF and DSS patients were observed to bemuch higher than in DHF and controls andan up regulation of intra-platelet L-argininenitric oxide pathway was observed in denguepatients and this was associated with thedecrease in platelet aggregation- a possiblereason to why thrombocytopenia occurs indengue. Currently, investigations into the roleof oxidative stress in dengue infection of themicrovascular ECs are being undertaken incollaboration with the University of Hong Kong.

Similarly, many other viruses have been knownto target the endothelium leading enhancedpermeability- hence we are also investigatingendothelium dysfunctions caused by otherviruses including respiratory viruses, influenzavirus, and neuroviruses. By gaining an under-standing of the mechanism of endotheliumdysfunction in infectious disease, we hope tomove on to finding therapeutic strategies toprevent/limit/reverse the disruption of theendothelial layer and thus minimise the severityof disease.

Antimicrobial/Antiviral DrugDevelopment

Dengue

The burden of dengue infection is consistentlyincreasing as there is no licensed vaccine orapproved antiviral drugs to combat DENVinfection. At present, the management of thisdisease consists primarily of supportive caresuch as administration of antipyretics,analgesics and resuscitation with intravenousfluid to restore the lost fluid in patientsmanifesting vascular leakage. Considering thehigh mortality and morbidity rates due to DENVinfection, several research groups in academiaand industry are involved in the developmentand identification of specific vaccine candidates,and novel therapeutics to prevent dengue.The three major approaches being pursued tocontrol dengue infections are vector control,dengue vaccines and antiviral therapy.Currently, the only method that is available toprevent this disease is vector control despitebeing expensive, ineffective and difficult tosustain. Several dengue vaccine candidates arebeing tested at different stages of pre-clinicalor clinical development but, so far none have

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Yeo A S, Azhar N A, Yeow W, Talbot Jr C C, Khan M A, Shankar E M, and Sekaran S D (2014)

Appanna R, Wang S M, Ponnampalavanar S A, Lum L C S, and Devi Sekaran S (2012)

been approved for clinical use. Hence, specificantiviral therapy needs to be developed for thetreatment of DENV infection. Our past andcurrent projects include (i) the application ofsiRNA to inhibit entry of dengue virus intotarget cells; (ii) use of natural products such asPhyllanthus plant species to inihibit denguevirus infections; and (iii) using combinatorialbioinformatics to design antimicrobialpeptides (AMPs) against dengue virus.

Dengue virus-host cell interaction is initiatedwhen the virus binds to the attachment receptors followed by endocytic internalisationof the virus particle. Successful entry into thecell is necessary for infection initiation; hencesilencing of GRP78 and clathrin-mediatedendocytosis was conducted to evaluate itseffect on dengue virus entry and multiplicationinto HepG2 cells while silencing of CD-14associated molecule and clathrin-mediatedendocytosis was done to assess viral entryinto monocytes. In both cell types, we showedthat indeed silencing the attachment receptor,clathrin-mediated endocytosis genes, cellsurface receptors and clathrin mediatedendocytosis genes using RNA interferencecould inhibit dengue virus entry and multipli-cation into HepG2 cells – leading to reductionof infected cells as well as the viral load, whichmight function as a unique and promisingtherapeutic agent for attenuating dengueinfection and prevent the development ofdengue fever to the severe life-threateningDHF or DSS.

Besides the application of siRNAs, we alsoattempted to evaluate the possibility of naturalproducts such as Phyllanthus (Euphorbiaceae),a local medicinal plant also known as DukungAnak as a antiviral drug agent. In Ayurvedicmedicine, Phyllanthus has a long tradition ofuse to treat jaundice, gonorrhea, frequentmenstruation, dysentery and diabetes as wellas skin ulcers, sores, swelling, and itchiness.In traditional Chinese medicine, Phyllanthushas been used for generations to eliminategallstones and kidney stones, as well as animmune system stimulator. We demonstratedthat a cocktail of extracts consisting of fourspecies of Phyllanthus (P.amarus, P.niruri,P.urinaria, and P.watsonii) had inhibitoryactivity against DENV2 with more than 90%of virus reduction in simultaneous treatment.Two-dimensional analysis revealed significantlyaltered levels of thirteen proteins which wereinvolved in several biological processes,including viral entry, viral transcription andtranslation regulations, cytoskeletal assembly,and cellular metabolisms.

The design of novel therapeutic approaches invarious studies has focused on targeting thedifferent stages of the viral replication cycle.This has been accomplished in in vitro studiesby the use of a number of inhibitory agentsdirected against a wide range of both cellularand viral targets. Small-molecule inhibitorssuch as Ivermectin, Mycophenolic acid andBrequinar, siRNAs and antiviral peptides havebeen designed and tested for their antiviraleffects. The peptide- or protein-basedtherapeutics are of interest primarily becausethey bind with exquisite specificity to their in vivo targets, resulting in exceptionally highpotencies of action and relative few off-targetside-effects. Synthetic AMPs are believed tobehave similarly with natural AMPs present inthe body being potent and broad spectrum;however working mostly like immunomodulators.The application of AMPs is a promising futurestrategy against microbial infections especiallywith the augmented drug resistances inmicrobes. Despite the generic disadvantages ofpeptides such as their low systemic stability,high clearance, poor membrane permeability,

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Alhoot M A, Rathinam A K, Wang S M, Manikam R, and Sekaran S D (2013)

more than 100 peptide-based drugs are inclinical use for the treatment of diseases suchas multiple sclerosis, breast cancer, prostatecancer and so on. One of our major interests isto develop novel peptides as antivirals againstDENV. The peptides exhibiting antiviral effectsby inducing conformational changes in the Eprotein or by inhibiting the E-PrM proteininteractions have been reported by us as well asby others. Currently, the effect of peptides onDENV replication by targeting its non-structuralproteins is being further examined. The non-structural proteins (NS1 to NS5) playcrucial roles in the viral replication and hence,in the pathogenesis of dengue infection.Therefore, our interest lies in designing novelpeptides targeting the non-structural proteinsof DENV and testing their potencies ininhibiting DENV replication.

Pneumococcal diseases

Pneumococcal diseases represent a global threatheavily affecting children aged less than twoand the elderly adults caused by Streptococcuspneumoniae or pneumococcus, a Gram-positive,alpha-hemolytic, aerotolerant anaerobic memberof the genus Streptococcus. This pathogen wasa major cause of pneumonia in the late 19thcentury and represents one of the major

etiological agents causing life-threateningdiseases such as pneumonia, meningitis, andbacteremia. Although antimicrobial drugssuch as penicillin have diminished the riskfrom pneumococcal disease, the proportion ofstrains that are resistant to antibiotics is steadilyincreasing. Moreover, the high prevalence ofantibiotic-resistant pneumococci has greatlylimited the choice of empirical therapy andthus the treatment outcomes. Therefore, animproved treatment and/or vaccine againstS. pneumoniae are one of the top vaccine priorities in the world. Certain proteins orenzymes displayed on the surface of S. pneumoniae significantly contribute topathogenesis and might be involved in thedisease process caused by these pathogens.

Antimicrobial peptides (AMPs) represent animportant part of the innate immune systemand produced by virtually all living species todefend themselves against microbial pathogens.These peptides are typically short positivelycharged, amphiphilic, and have been isolatedfrom single-celled microorganisms up tohuman beings. The primary role of thesepeptides is to kill of invading pathogenicorganisms. It was formerly proposed thatpermeabilisation of the bacterial cell membranewas the only mode of action of antibacterial

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Lee S H, Tang Y Q, Rathkrishnan A, Wang S M, Ong K C, Manikam R, and Sekaran S D (2013)

peptides; however, several studies haverevealed that some antibacterial peptidestranslocate into cells and do not cause membranepermeabilisation but rather mediate bacterialcell death by targeting essential intracellularprocesses. Because of their broad-spectrumactivity, AMPs are considered to be verypromising candidates as novel antimicrobialagents. Our main focus is to design novelsynthetic antimicrobial peptides (AMPs) aspotential antimicrobial candidates againstStreptococcus pneumoniae. At the presentstage, we have designed and tested a series ofhybrid AMPs exhibiting strong antipneumococ-cal effects of which DM3 possessed promisingin vitro and in vivo antipneumococcal activitiesand this has been patented. Based on this currentexperience, we have begun to investigate otherhybrids generated based on Ranalexin andIndolicidin which showed potent antipneumo-coccal activity and the mechanisms of action ofthese AMPs will be evaluated alongside in vivotherapeutic efficacy.

Anti-cancer drug development

In conjunction with Professor Shamala’s ambition in search of therapeutic agentsagainst microbes, we had previously venturedinto looking at the anti-cancer properties ofthe local plant known as Phyllanthus. We havedemonstrated that Phyllanthus possessedbroad spectrum anticancer properties as itexhibited anti-proliferative, anti-metastaticand anti-angiogenic effects as well aspossesses capability to induce apoptosis in anumber of human cancers cell lines includingbreast, lung, prostate and melanoma. Many ofits anti-cancer actions were associated withdisruption of multiple survival pathways andprotein expression in Phyllanthus treatedcancer cells. This project has resulted in 5publications in peer-reviewed journals and 1book chapter.

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Tang Y Q, Jaganath I, Manikam R, and Sekaran S D (2013)

Prof. Dr. Shamala Devi A/P K.C Sekaran

Department of Medical Microbiology

Faculty of Medicine

Tel: 60379675759

shamala@um.edu.mywww.medicine.um.edu.my

Funding Bodies :University of Malaya (Postgraduate Research Fund, High ImpactResearch Grants), Ministry of Higher Learning (MOHE), Ministry ofScience & Technology Malaysia (IRPA RM6, MOSTI RM9, Brain GainMalaysia, ScienceFund, Scientific Advancement Grant Allocation (SAGA),Exploratory Research Grant Scheme (ERGS), Fundamental ResearchGrant Scheme (FRGS)) and Malaysian Agricultural Research and

Development Institute (MARDI) and TDR/WHO.

Collaborators : TDR/WHO, The John Hopkins University, Perdana University GraduateSchool of Medicine Malaysia, University of South Florida, Colorado StateUniversity, University of Tubingen, Institute for Medical ResearchMalaysia, Faculty of Biomedical Engineering University of Malaya, University Sains Malaysia and the University of Brunei.