REVIEW ARTICLEAutoimmunity in dengue pathogenesisShu-Wen Wan a,b, Chiou-Feng Lin a,b,c,d, Trai-Ming Yeh b,c,e,Ching-Chuan Liu b,f, Hsiao-Sheng Liu a,b,c, Shuying Wang a,b,c, Pin Ling a,b,c,Robert Anderson a,b,g,h,i, Huan-Yao Lei a,b,c,j, Yee-Shin Lin a,b,c,*aDepartment of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, TaiwanbCenter of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, TaiwancInstitute of Basic Medical Sciences, National Cheng Kung University Medical College, Tainan, TaiwandInstitute of Clinical Medicine, National Cheng Kung University Medical College, Tainan, TaiwaneDepartment of Medical Laboratory Science and Biotechnology, National Cheng Kung University Medical College, Tainan,TaiwanfDepartment of Pediatrics, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, TaiwangDepartment of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, CanadahDepartment of Pediatrics, Dalhousie University, Halifax, Nova Scotia, CanadaiCanadian Center for Vaccinology, Dalhousie University, Halifax, Nova Scotia, CanadaReceived 20 October 2012; accepted 9 November 2012KEYWORDSautoimmunity;dengue;immunopathogenesisDengue is one of the most important vector-borne viral diseases. With climate change and theconvenience of travel, dengue is spreading beyond its usual tropical and subtropical bound-aries. Infection with dengue virus (DENV) causes diseases ranging widely in severity, fromself-limited dengue fever to life-threatening dengue hemorrhagic fever and dengue shocksyndrome. Vascular leakage, thrombocytopenia, and hemorrhage are the major clinical mani-festations associated with severe DENV infection, yet the mechanisms remain unclear. Besidesthe direct effects of the virus, immunopathogenesis is also involved in the development ofdengue disease. Antibody-dependent enhancement increases the efﬁciency of virus infectionand may suppress type I interferon-mediated antiviral responses. Aberrant activation of T cellsand overproduction of soluble factors cause an increase in vascular permeability. DENV-induced autoantibodies against endothelial cells, platelets, and coagulatory molecules leadto their abnormal activation or dysfunction. Molecular mimicry between DENV proteins andhost proteins may explain the cross-reactivity of DENV-induced autoantibodies. Although no* Corresponding author. Department of Microbiology and Immunology, National Cheng Kung University Medical College, 1 University Road,Tainan 701, Taiwan.E-mail address: firstname.lastname@example.org (Y.-S. Lin).jDr Huan-Yao Lei passed away during the preparation of this manuscript. This review article is dedicated to Dr Lei.0929-6646/$ - see front matter Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.http://dx.doi.org/10.1016/j.jfma.2012.11.006Available online at www.sciencedirect.comjournal homepage: www.jfma-online.comJournal of the Formosan Medical Association (2013) 112, 3e11
licensed dengue vaccine is yet available, several vaccine candidates are under development.For the development of a safe and effective dengue vaccine, the immunopathogenic compli-cations of dengue disease need to be considered.Copyright ª 2012, Elsevier Taiwan LLC & Formosan Medical Association. All rights reserved.IntroductionDengue virus (DENV) belongs to the genus Flavivirus of thefamily Flaviviriade. Based on neutralization assay data,four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) canbe distinguished. DENV is transmitted to humans mainly byAedes aegypti and Aedes albopictus.1About 50 milliondengue infection cases, with around 500,000 cases per yearof severe dengue, have mainly been reported in the Asia-Paciﬁc region, the Americas, and Africa. All four DENVserotypes are now circulating in these areas.2The trans-mission efﬁciency and disease expression between theserotypes are still uncertain, but DENV-2 and DENV-3 mightcontribute the most to disease severity and mortality.3There have been several major outbreaks of dengue inTaiwan, particularly in 1981, 1987e1988, 2001e2002, and2007. Dengue outbreaks involve various combinations ofdengue serotypes, with certain serotypes predominating,such as DENV-2 in the year 2002.4,5Recent reports haveclariﬁed the usual pattern in Taiwan outbreaks: starting byimport from abroad in early summer, spreading out locally,and ending in the winter. Dengue is primarily an adultdisease in Taiwan. Most cases of dengue fever (DF) havebeen reported in individuals in the 50e54-year age rangeand most cases of dengue hemorrhagic fever (DHF) in the60e64-year range.4However, dengue usually occurs inchildren in hyperendemic Southeast Asia. Secondaryinfection of DENV-2 was prevalent in the year 2002, butprimary infection of DENV-1 or DENV-3 in 2004e2007. Inaddition, adults or the elderly have a greater risk ofdeveloping the severe dengue disease.4DENV is a lipid-enveloped, single-positive-RNA virus,with a genome of about 10.7 kb. RNA of the virus is trans-lated to three structural proteins, namely capsid protein(C), precursor membrane protein (prM), and envelopeprotein (E). Besides the structural proteins, there are sevennonstructural proteins (NS), which are involved in variousfunctions affecting viral replication and disease pathogen-esis.6,7The replication cycle of DENV begins when thevirions attach to the surface of host cells and subsequentlyenter the cells by receptor-mediated endocytosis. Acidiﬁ-cation of the endosomal vesicle triggers conformationalchanges in the virion, which results in the fusion of the viraland cell membranes. After the fusion has occurred, thenucleocapsid is released into the cytoplasm. The positive-sense RNA is translated into a single polyprotein that isprocessed cotranslationally and post-translationally by viraland host proteases. Genome replication occurs on intra-cellular membranes. Virus assembly occurs on the surfaceof the endoplasmic reticulum (ER) when the structuralproteins and the newly synthesized RNA bud into the lumenof ER. The virion is maturated in the Golgi compartmentand exits by the secretory pathway. Two processes areinvolved in virus maturation. First, the prM protein iscleaved by host furin and forms the M protein in the trans-Golgi network. Second, the E protein undergoes a majorconformational rearrangement during the maturation ofvirus particles during exocytosis.7,8Infection with DENV causes diseases ranging from mildDF to severe DHF and dengue shock syndrome (DSS). DHF/DSS usually occurs in patients who are secondarily infectedwith heterotypic DENV, but it also occurs in case of primaryinfection.9DF presents with an onset of fever accompaniedby severe headache, retro-orbital pain, myalgia, arthralgia,abdominal pain, rash, and minor hemorrhage in the form ofpetechiae, epistaxis, or gingival bleeding. Leukopenia isa common ﬁnding in laboratory tests, whereas thrombocy-topenia may occasionally be observed in DF patients.10Inaddition to all the symptoms of DF, DHF is characterized bysevere hemorrhage (positive tourniquet test or spontaneousbleeding), thrombocytopenia (platelet counts <100,000/mm3), plasma leakage (increased hemoconcentration orﬂuid effusion in chest or abdominal cavities), and hepato-megaly (elevation of serum transaminases). The WorldHealth Organization (WHO) classiﬁes DHF into four grades(IdIV). DHF grades I and II represent relatively mild caseswithout shock, whereas grades III and IV cases are moresevere and may lead to disseminated intravascular coagu-lation.11e13There has been a systematic literature reviewsummarizing the difﬁculties in applying the criteria for DHFin the clinical situation. For example, the positive tourni-quet test indicative of hemorrhagic manifestation does notsigniﬁcantly distinguish between DHF and DF. In addition,the incidences of major manifestations (hemorrhage,thrombocytopenia, and plasma leakage) observed in DHFpatients span a large range.13Accordingly, the WHO clas-siﬁcation system is currently being reconsidered to be moresuitable for clinical practice. The new guidelines includedengue without warning signs, dengue with warning signs,and severe dengue. From recent studies, 13.7% of denguecases could not be classiﬁed using the DF/DHF/DSS classi-ﬁcation, whereas only 1.6% could not be classiﬁed using therevised classiﬁcation.14Hence, assessments of the newclassiﬁcation are still continuing, and the potential imple-mentation of the revised classiﬁcation has been proposed.The pathogenic mechanisms in DHF/DSS are complicatedand not fully resolved. Several mechanisms are involved inthe pathogenesis of DHF/DSS progression, including viralpathogenesis and immunopathogenesis. Viral pathogenesisreﬂects the pathology directly caused by the virus, and issubject to serotypic or genotypic differences. In contrast,immunopathogenesis encompasses other factors involvingthe host immune response, which may be involved in thepathogenesis.15For example, during secondary infection,the critical phase of disease occurs when the viralburden declines. This has led to the suggestion that4 S.-W. Wan et al.
immunopathogenic mechanisms, such as the adaptiveimmune response, inﬂammatory mediators, and autoim-munity, are important in the pathogenesis of denguedisease. Such mechanisms play signiﬁcant roles in majormanifestations of DHF, including hemorrhage, thrombocy-topenia, plasma leakage, and hepatomegaly (summarizedin Fig. 1).15e17Viral pathogenesisVirus variationVirus variation indicates the capacity of a virus to producedisease in a host. In the case of dengue, genetic differencesamong DENV isolates contribute to the severity of denguedisease. There are four antigenically distinct serotypes ofDENV, each of which can cause an outbreak of denguedisease.2However, DENV-2 and DENV-3 may contribute themost to disease severity and mortality.3Viral genetic18e20and structural21differences have been shown to inﬂuencehuman disease severity. Recently, viral genetic differenceswere demonstrated to be a contributing factor to virulencein a mouse model.22However, it remains to be determinedwhether these serotypic or genotypic differences observedin vitro or in mouse models, respectively, contribute tovirulence differences in humans.Cell and tissue tropismCell and tissue tropism of DENV likely have a major impacton the outcome of DENV infection. Langerhans cells(dermal dendritic cells) are generally proposed to be theinitial target for DENV infection at the site of the mosquitobite,23followed by the systemic infection of macrophages/monocytes24and viral entry into the blood. From autopsiesof fatal cases, DENV has been found in the skin, liver,spleen, lymph node, kidney, bone marrow, lung, thymus,and brain.11Besides the primary targets (dendritic cells andmacrophages) of DENV, other potential target cellsincluding hepatocytes, endothelial cells, and neuronal cellshave been detected in mouse models. DENV can not onlyreplicate in these cells but also contribute to their damageand/or dysfunction. For example, mice inoculated withDENV by intraperitoneal,25intradermal,26,27or intra-cerebal28routes have been shown to display liverpathology, hemorrhagic or neurological symptoms. Eleva-tion of serum transaminases, hemorrhage, and fatalencephalitis have been observed in these mouse models,and provide mechanistic insights for the manifestations ofdengue disease.25e28The range of these cell or tissue typesinfected with DENV suggest that the receptors of DENV arediverse or broadly distributed. The afﬁnity of DENV withthose receptors might inﬂuence virus infectivity as well asvirulence. A single amino acid mutation on E protein of theﬂavivirus (Murray Valley encephalitis virus) have beendemonstrated to cause altered cell tropism, includingdifferences in entry kinetics, attachment to mammaliancells, and virulence in mice.29In summary, the factors thatdetermine the numbers and fates of infected cells atspeciﬁc sites likely contribute to the pathology of denguedisease.ImmunopathogenesisAntibody-dependent enhancementAntibody-dependent enhancement (ADE) is a well-knownhypothesis of dengue disease pathogenesis. Epidemiolog-ical evidence suggests that the presence of pre-existingsubneutralizing antibodies (Abs) is a major factor fordeveloping DHF/DSS in both infants and adults.30EnhancingAbs increase the efﬁciency of virus attachment andFigure 1 A hypothetical model of dengue pathogenesis. Viral and immunological factors contribute to clinical manifestations,including severe hemorrhage, thrombocytopenia, plasma leakage, and hepatomegaly. DENV Z dengue virus.Autoimmunity in dengue 5
internalization through Fcg receptor (FcgR)-dependent30orFcgR-independent mechanisms.31Enhancing Abs alsocontribute to the binding of DENV to platelets.32Recently, a new hypothesis (termed intrinsic ADE)postulates that FcgR-mediated DENV internalizationsuppresses the type I interferon (IFN)-mediated antiviralresponses by inhibiting antiviral genes and enhancinginterleukin-10 (IL-10) production, which suppresses theIFN-g signaling pathway and promotes T-helper-2responses.33e35T-helper-1 responses are required for virusclearance; however, T-helper-2 responses have limitedantiviral effect and enhance the production of Abs. Thismay lead to high levels of both viral loads and Abs in denguepatients. Besides the ampliﬁcation of viral output, ADEenhances cytokine and chemokine production,36e39cellapoptosis,40and tumor necrosis factor-a (TNF-a)-mediatedendothelial cell activation.36,41Cellular immune responseAlthough memory T cells, which cross-react with heterolo-gous viruses, can provide partial protective immunity, theymay cause immunopathology.42According to the “originalantigenic sin” model, low-afﬁnity memory T cells generatedduring primary DENV infection expand selectively duringthe secondary infection of another virus serotype, prior tothe activation of naı¨ve T cells of higher avidity for thesecond DENV serotype. The cross-reactive T cells producehigh concentrations of inﬂammatory cytokines and maycontribute to the pathogenesis of plasma leakage in denguedisease.11,43e45DENV-speciﬁc human CD4þcytotoxic T cellshave been demonstrated to lyse bystander target cellsin vitro.46This mechanism may provide an explanation forlymphocyte activation and hepatocyte damage in a DENV-infected mouse model.47A recent study demonstratedthat regulatory T-cell frequencies and regulatory T-cell/effector T-cell ratios are increased in acute dengueinfection.48Soluble factorsSeveral studies have indicated that the concentrations ofcytokines, chemokines, or other mediators might besigniﬁcantly increased during DENV infection. Higher levelsof IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-18, monocyte che-moattractant protein-1 (MCP-1), macrophage migrationinhibitory factor (MIF), transforming growth factor-b, TNF-a, and IFN-g have been found in the plasma of severedengue patients.43,49e58These mediators play central rolesin regulating the immune response to dengue. In particular,TNF-a produced by dengue-infected monocytes36as well asby mast cells41triggers the activation of vascular endo-thelial cells. Also, some studies demonstrated that TNF-a contributes to endothelial permeability and hemorrhageduring DENV infection in animal models.26,59In addition toTNF-a,60several studies demonstrated that IL-8,61MCP-1,57MIF,62and metalloproteinase 963,64promoted increasedendothelial permeability in vitro. Furthermore, IL-6 and IL-8 have been found to be associated with the activation ofcoagulation and ﬁbrinolysis.65e67IL-8 levels have been re-ported to be increased in most dengue patients andcorrelated with degranulation of neutrophils.68In addition,levels of IL-10 have been shown to correlate with the loss ofplatelets and failure of platelet function.69High levels of C3a and C5a have been detected in thesera from severely affected dengue patients.70,71C3a andC5a, the products of C3 and C5 cleavage, are anaphylo-toxins, which promote chemotoxis of immune cells andcontribute to inﬂammatory responses. Soluble NS1 andanti-DENV Abs have also been reported to activatecomplement, by binding on the surface of infected endo-thelial cells.70,72High plasma levels of NS1 and terminalcomplement complex have been detected in DENV-infectedpatients, and these were correlated with vascular leakageas well as disease severity.70High levels of regulatoryfactors D and H have also been reported in DHF patientscompared to those in DF patients. The imbalance of factorsD and H caused alternative complement pathway deregu-lation and might correlate with disease severity.72AutoimmunityAutoimmunity and molecular mimicry have been demon-strated in various viral infections, such as Coxsackievirusand EpsteineBarr virus, and have been implicated in humanautoimmune diseases.73Autoantibodies represent anotherimportant factor involved in dengue disease pathogenesis.Several studies showed that the generation of autoanti-bodies against platelets,74e76endothelial cells,77,78andcoagulatory molecules77e81was associated with denguedisease. Molecular mimicry between platelets, endothelialcells, and coagulatory molecules with NS1, prM, and Eproteins may explain the cross-reactivity of anti-NS1, anti-prM, and anti-E Abs, respectively, to host proteins. Theconsequences of these cross-reactive Abs are plateletdysfunction, endothelial cell apoptosis, coagulation defect,and macrophage activation.73,82A schematic model ofimportant dengue manifestations induced by cross-reactiveautoantibodies is illustrated in Fig. 2.Our studies showed that the levels of antiplatelet andantiendothelial cell autoantibodies are higher in the sera ofDHF/DSS patients than in that of DF patients. Immuno-globulin M (IgM) present in the sera of DHF patients playeda more dominant role than IgG in the cross-reactivity withplatelets and endothelial cells. Absorption experimentsrevealed that anti-DENV NS1 Abs in patients’ sera areresponsible for the cross-reactivity, resulting in plateletdysfunction and endothelial cell apoptosis.74,78,83Theseﬁndings suggest that DENV-induced antoantibodies might beassociated with thrombocytopenia and plasma leakage.Anti-DENV NS1 Abs, which were generated from mice,cross-reacted with endothelial cells and triggered apoptosisby nitric oxide production.84In addition, anti-DENV NS1 Absinduced endothelial cells to express IL-6, IL-8, MCP-1, andintercellular adhesion molecule-1. The activation of endo-thelial cells by anti-DENV NS1 Abs demonstrated theinvolvement of anti-DENV NS1 Abs in the vasculopathy ofDENV infection.85Furthermore, mice actively immunizedwith NS1 proteins or passively administrated with anti-DENVNS1 Abs showed a hepatitis-like pathologic effect. Theseresults revealed that anti-DENV NS1 Abs might play a role inliver damage, which is an important manifestation of6 S.-W. Wan et al.
dengue disease.86From proteomic analysis, the potentialcandidate proteins on endothelial cells, recognized by anti-DENV NS1 Abs, include ATP synthase b-chain, vimentin,heat shock protein 60, and protein disulﬁde isomerase. TheC-terminal amino acid (a.a.) 311e352 region of DENV NS1shows certain degrees of homology with the candidateproteins.87Protein disulﬁde isomerase was recognized byanti-DENV NS1 Abs both on endothelial cells and on plate-lets.87,88We also found that the C-terminal region of NS1was responsible for cross-reactivity with platelets. Thedeletion of C-terminal region (a.a. 277e352) of NS1 abol-ished anti-NS1-mediated platelet aggregation and bleedingtendency.89These results suggest a mechanism of molec-ular mimicry in which Abs against DENV NS1 cross-reactwith endothelial cells and platelets.Previous studies in our laboratory identiﬁed importantcross-reactive epitopes on the C terminus (a.a. 271e352) ofDENV NS1 proteins.87e90Recent studies also indicated thata.a. 116e119 of DENV NS1 shared sequence similarity withhuman LYRIC protein (lysine-rich CEACAM1 co-isolated) a.a.334e337.82Furthermore, despite the absence of an argi-nineeglycineeaspartic acid (RGD) motif in the DENV NS1protein sequence, RGD structural mimicry exists within theNS1 protein. Since RGD is an important motif for matrix-integrin-mediated cell adhesion, anti-NS1 Abs could blockRGD-mediated cell adhesion.91These ﬁndings suggest theexistence of still other cross-reactive epitopes, whichshould be investigated in the future.Besides thrombocytopenia and plasma leakage,abnormal coagulopathy can also be observed in severedengue patients. Hemostatic parameters altered in DHF/DSS include prolonged thrombin time and activated partialthromboplastic time, decreased levels of ﬁbrinogen, andincreased levels of ﬁbrinogen degradation products.92Several studies suggested that autoantibodies may partici-pate in abnormal hemostasis during DENV infection. Absagainst NS1 and E proteins have been shown to cross-reactwith human blood coagulation factors, ﬁbrinogen, andplasminogen.77,79,80By sequence alignment, DENV proteins,including core, E, prM, and NS1, have shown differentlevels of sequence similarity with different coagulatory-associated molecules such as factor X, factor XI, and plas-minogen.73Although the effects of these autoantibodies oncoagulatory factors are still unclear, some reports demon-strated that DENV-induced autoantibodies might interferewith human ﬁbrinolysis.93,94In our previous studies, the titers of DENV-inducedautoantibodies reached peak levels in the acute phase,declined during the convalescent stage, and lasted forseveral months.74,78This time course is different fromchronic virus infection-associated autoimmune disease.73A recent case report showed a dengue patient withnumerous autoimmune features,95and another reportshowed a dengue patient in whom dengue evolved intosystemic lupus erythematous and lupus nephritis aftera month.96A follow-up study reported that dengue-infected individuals have long-term persistence of clin-ical symptoms with complement factors, rheumatoidfactor, C-reactive protein, antinuclear Abs, and immunecomplexes.97From these studies, it appears that DENVinfection may trigger abnormal immune responsescausing autoimmune reactions. Therefore, autoimmuneFigure 2 A schematic model of autoantibody-mediated immunopathogenesis in DENV infection. Molecular mimicry betweenplatelets, endothelial cells, and coagulatory molecules with NS1, prM, E, and C proteins underlies the cross-reactivity of anti-NS1,anti-prM, anti-E, and anti-C Abs, respectively, to host proteins. Abs Z antibodies; C Z capsid protein; DENV Z dengue virus;E Z envelope protein; NS Z nonstructural protein; prM Z precursor membrane protein.Autoimmunity in dengue 7
complications should be considered when developinga safe dengue vaccine.Dengue vaccine strategyAlthough no licensed dengue vaccine is yet available,several vaccine candidates are under development.Although live viral vaccines have advanced to clinical trials,they encountered new difﬁculties, such as viral interfer-ence among the four serotypes in tetravalent formulations.For safety concerns, nonviral vaccines have also beendeveloped, particularly subunit vaccines mostly focused onthe E protein or its derivatives. However, the challenge ofeliciting balanced levels of neutralizing Abs to each of thefour viral serotypes remains a major concern.12,98NS1 is not a virion-associated protein, and anti-NS1 Absdo not enhance DENV infection. Anti-DENV NS1 Abs ﬁxcomplement and trigger complement-mediated lysis ofDENV-infected cells.99Previous studies showed that activeimmunization with NS1 proteins and passive immunizationwith anti-NS1 Abs could provide protection to mice againstDENV challenge.99,100However, anti-NS1 Abs still showsome pathogenic effects both in vitro and in vivo.15,73Further mapping and/or genetic manipulation of the rele-vant pathogenic epitopes will be important for the devel-opment of a safe dengue NS1 vaccine.ConclusionsDengue is one of the most important vector-borne viraldiseases in the world. The complexity of dengue immuno-pathogenesis increases the difﬁculties associated with thedevelopment of a dengue vaccine. A successful denguevaccine must be effective against all four serotypes, avoidpotential ADE-associated pathogenic effects, as well as befree of potential autoimmune complications.AcknowledgmentsThis work was supported by grants NSC100-2321-B006-004and NSC100-2325-B006-007 from the National ScienceCouncil, Taiwan; NHRI-100A1-PDCO-0209115 from theNationalHealth Research Institutes,Taiwan; andDOH101-TD-B-111-002 from the Multidisciplinary Center of Excellence forClinical Trial and Research, Department of Health, Taiwan.References1. Monath TP. Dengue: the risk to developed and developingcountries. Proc Natl Acad Sci U S A 1994;91:2395e400.2. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J,Gubler DJ, et al. Dengue: a continuing global threat. Nat RevMicrobiol 2010;8:S7e16.3. Guzman A, Isturiz RE. Update on the global spread of dengue.Int J Antimicrob Agents 2010;36(Suppl. 1):S40e2.4. Lin CC, Huang YH, Shu PY, Wu HS, Lin YS, Yeh TM, et al.Characteristic of dengue disease in Taiwan: 2002e2007. Am JTrop Med Hyg 2010;82:731e9.5. Chang SF, Huang JH, Shu PY. Characteristics of dengueepidemics in Taiwan. J Formos Med Assoc 2012;111:297e9.6. Henchal EA, Putnak JR. The dengue viruses. Clin MicrobiolRev 1990;3:376e96.7. Clyde K, Kyle JL, Harris E. Recent advances in decipheringviral and host determinants of dengue virus replication andpathogenesis. J Virol 2006;80:11418e31.8. Rodenhuis-Zybert IA, Wilschut J, Smit JM. Dengue virus lifecycle: viral and host factors modulating infectivity. Cell MolLife Sci 2010;67:2773e86.9. Gubler DJ. Dengue and dengue hemorrhagic fever. ClinMicrobiol Rev 1998;11:480e96.10. Kittigul L, Pitakarnjanakul P, Sujirarat D, Siripanichgon K. Thedifferences of clinical manifestations and laboratory ﬁndingsin children and adults with dengue virus infection. J Clin Virol2007;39:76e81.11. Martina BE, Koraka P, Osterhaus AD. Dengue virus pathogen-esis: an integrated view. Clin Microbiol Rev 2009;22:564e81.12. Murphy BR, Whitehead SS. Immune response to dengue virusand prospects for a vaccine. Annu Rev Immunol 2011;29:587e619.13. Bandyopadhyay S, Lum LCS, Kroeger A. Classifying dengue:a review of the difﬁculties in using the WHO case classiﬁca-tion for dengue haemorrhagic fever. Trop Med Int Health2006;11:1238e55.14. Barniol J, Gaczkowski R, Barbato EV, da Cunha RV, Salgado D,Martinez E, et al. Usefulness and applicability of the reviseddengue case classiﬁcation by disease: multi-centre study in 18countries. BMC Infect Dis 2011;11:106e17.15. Whitehorn J, Simmons CP. The pathogenesis of dengue.Vaccine 2011;29:7221e8.16. Lin CF, Wan SW, Cheng HJ, Lei HY, Lin YS. Autoimmunepathogenesis in dengue virus infection. Viral Immunol 2006;19:127e32.17. Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH, Liu CC. Immunopa-thogenesis of dengue virus infection. J Biomed Sci 2001;8:377e88.18. Pandey BD, Morita K, Hasebe F, Parquet MC, Igarashi A.Molecular evolution, distribution and genetic relationshipamong the dengue 2 viruses isolated from different clinicalseverity. Southeast Asian J Trop Med Public Health 2000;31:266e72.19. Vaughn DW, Green S, Kalayanarooj S, Innis BL,Nimmannitya S, Suntayakorn S, et al. Dengue viremia titer,antibody response pattern, and virus serotype correlate withdisease severity. J Infect Dis 2000;181:2e9.20. Cologna R, Rico-Hesse R. American genotype structuresdecrease dengue virus output from human monocytes anddendritic cells. J Virol 2003;77:3929e38.21. Leitmeyer KC, Vaughn DW, Watts DM, Salas R, Villalobos I,de C, et al. Dengue virus structural differences that correlatewith pathogenesis. J Virol 1999;73:4738e47.22. Prestwood TR, Prigozhin DM, Sharar KL, Zellweger RM,Shresta S. A mouse-passaged dengue virus strain with reducedafﬁnity for heparan sulfate causes severe disease in mice byestablishing increased systemic viral loads. J Virol 2008;82:8411e21.23. Wu SJ, Grouard-Vogel G, Sun W, Mascola JR, Brachtel E,Putvatana R, et al. Human skin Langerhans cells are targets ofdengue virus infection. Nat Med 2000;6:816e20.24. Kou Z, Quinn M, Chen H, Rodrigo WW, Rose RC, Schlesinger JJ,et al. Monocytes, but not T or B cells, are the principal targetcells for dengue virus (DV) infection among human peripheralblood mononuclear cells. J Med Virol 2008;80:134e46.25. Paes MV, Lenzi HL, Nogueira AC, Nuovo GJ, Pinhao AT,Mota EM, et al. Hepatic damage associated with dengue-2virus replication in liver cells of BALB/c mice. Lab Invest2009;89:1140e51.26. Chen HC, Hofman FM, Kung JT, Lin YD, Wu-Hsieh BA. Bothvirus and tumor necrosis factor alpha are critical for8 S.-W. Wan et al.
endothelium damage in a mouse model of dengue virus-induced hemorrhage. J Virol 2007;81:5518e26.27. Yen YT, Chen HC, Lin YD, Shieh CC, Wu-Hsieh BA. Enhance-ment by tumor necrosis factor alpha of dengue virus-inducedendothelial cell production of reactive nitrogen and oxygenspecies is key to hemorrhage development. J Virol 2008;82:12312e24.28. Despres P, Frenkiel MP, Ceccaldi PE, Duarte Dos Santos C,Deubel V. Apoptosis in the mouse central nervous system inresponse to infection with mouse-neurovirulent dengueviruses. J Virol 1998;72:823e9.29. Lee E, Lobigs M. Substitutions at the putative receptor-binding site of an encephalitic ﬂavivirus alter virulence andhost cell tropism and reveal a role for glycosaminoglycans inentry. J Virol 2000;74:8867e75.30. Halstead SB. Neutralization and antibody-dependentenhancement of dengue viruses. Adv Virus Res 2003;60:421e67.31. Huang KJ, Yang YC, Lin YS, Huang JH, Liu HS, Yeh TM, et al.The dual-speciﬁc binding of dengue virus and target cells forthe antibody-dependent enhancement of dengue virusinfection. J Immunol 2006;176:2825e32.32. Wang S, He RT, Patarapotikul J, Innis BL, Anderson R. Anti-body-enhanced binding of dengue virus to human platelets.Virology 1995;213:254e7.33. Ubol S, Phuklia W, Kalayanarooj S, Modhiran N. Mechanisms ofimmune evasion induced by a complex of dengue virus andpreexisting enhancing antibodies. J Infect Dis 2010;201:923e35.34. Ubol S, Halstead SB. How innate immune mechanismscontribute to antibody-enhanced viral infections. ClinVaccine Immunol 2010;17:1829e35.35. Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM.Intrinsic antibody-dependent enhancement of microbialinfection in macrophages: disease regulation by immunecomplexes. Lancet Infect Dis 2010;10:712e22.36. Anderson R, Wang S, Osiowy C, Issekutz AC. Activation ofendothelial cells via antibody-enhanced dengue virus infec-tion of peripheral blood monocytes. J Virol 1997;71:4226e32.37. King C, Anderson R, Marshall JS. Dengue virus selectivelyinduces human mast cell chemokine production. J Virol 2002;76:8408e19.38. Brown MG, King CA, Sherren C, Marshall JS, Anderson R. Adominant role for FcgRII in antibody-enhanced dengue virusinfection of human mast cells and associated CCL5 release. JLeuk Biol 2006;80:1242e50.39. Brown MG, McAlpine SM, Huang YY, Haidl I, Al-Aﬁf A,Marshall JS, et al. RNA sensors enable human mast cell anti-viral chemokine production and IFN-mediated protection inresponse to antibody-enhanced dengue virus infection. PLoSOne 2012;7:e34055.40. Brown MG, Huang YY, Marshall JS, King CA, Hoskin DW,Anderson R. Dramatic caspase-dependent apoptosis inantibody-enhanced dengue virus infection of human mastcells. J Leukoc Biol 2009;85:71e80.41. Brown MG, Hermann LL, Issekutz AC, Marshall JS, Rowter D,Al-Aﬁf A, et al. Dengue virus infection of mast cells triggersendothelial cell activation. J Virol 2011;85:1145e50.42. Selin LK, Varga SM, Wong IC, Welsh RM. Protective heterolo-gous antiviral immunity and enhanced immunopathogenesismediated by memory T cell populations. J Exp Med 1998;188:1705e15.43. Kurane I, Innis BL, Nimmannitya S, Nisalak A, Meager A,Janus J, et al. Activation of T lymphocytes in dengue virusinfections. High levels of soluble interleukin 2 receptor,soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. J Clin Invest 1991;88:1473e80.44. Mangada MM, Rothman AL. Altered cytokine responses ofdengue-speciﬁc CD4þT cells to heterologous serotypes. JImmunol 2005;175:2676e83.45. Hatch S, Endy TP, Thomas S, Mathew A, Potts J, Pazoles P,et al. Intracellular cytokine production by dengue virus-speciﬁc T cells correlates with subclinical secondary infec-tion. J Infect Dis 2011;203:1282e91.46. Gagnon SJ, Ennis FA, Rothman AL. Bystander target cell lysisand cytokine production by dengue virus-speciﬁc human CD4(þ)cytotoxic T-lymphocyte clones. J Virol 1999;73:3623e9.47. Chen HC, Lai SY, Sung JM, Lee SH, Lin YC, Wang WK, et al.Lymphocyte activation and hepatic cellular inﬁltration inimmunocompetent mice infected by dengue virus. J Med Virol2004;73:419e31.48. Luhn K, Simmons CP, Moran E, Dung NT, Chau TN, Quyen NT,et al. Increased frequencies of CD4þCD25highregulatory Tcells in acute dengue infection. J Exp Med 2007;204:979e85.49. Hober D, Poli L, Roblin B, Gestas P, Chungue E, Granic G, et al.Serum levels of tumor necrosis factor-alpha (TNF-alpha),interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta) indengue-infected patients. Am J Trop Med Hyg 1993;48:324e31.50. Chaturvedi UC. Shift to Th2 cytokine response in denguehaemorrhagic fever. Indian J Med Res 2009;129:1e3.51. Raghupathy R, Chaturvedi UC, Al-Sayer H, Elbishbishi EA,Agarwal R, Nagar R, et al. Elevated levels of IL-8 in denguehemorrhagic fever. J Med Virol 1998;56:280e5.52. Green S, Vaughn DW, Kalayanarooj S, Nimmannitya S,Suntayakorn S, Nisalak A, et al. Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity. JMed Virol 1999;59:329e34.53. Mustafa AS, Elbishbishi EA, Agarwal R, Chaturvedi UC.Elevated levels of interleukin-13 and IL-18 in patients withdengue hemorrhagic fever. FEMS Immunol Med Microbiol2001;30:229e33.54. Agarwal R, Elbishbishi EA, Chaturvedi UC, Nagar R,Mustafa AS. Proﬁle of transforming growth factor-beta 1 inpatients with dengue haemorrhagic fever. Int J Exp Pathol1999;80:143e9.55. Hober D, Nguyen TL, Shen L, Ha DQ, Huong VT, Benyoucef S,et al. Tumor necrosis factor alpha levels in plasma and whole-blood culture in dengue-infected patients: relationshipbetween virus detection and pre-existing speciﬁc antibodies.J Med Virol 1998;54:210e8.56. Chaturvedi UC, Agarwal R, Elbishbishi EA, Mustafa AS. Cyto-kine cascade in dengue hemorrhagic fever: implications forpathogenesis. FEMS Immunol Med Microbiol 2000;28:183e8.57. Lee YR, Liu MT, Lei HY, Liu CC, Wu JM, Tung YC, et al. MCP-1,a highly expressed chemokine in dengue haemorrhagicfever/dengue shock syndrome patients, may cause perme-ability change, possibly through reduced tight junctions ofvascular endothelium cells. J Gen Virol 2006;87:3623e30.58. Chen LC, Lei HY, Liu CC, Shiesh SC, Chen SH, Liu HS, et al.Correlation of serum levels of macrophage migration inhibi-tory factor with disease severity and clinical outcome indengue patients. Am J Trop Med Hyg 2006;74:142e7.59. Shresta S, Sharar KL, Prigozhin DM, Beatty PR, Harris E. Murinemodel for dengue virus-induced lethal disease with increasedvascular permeability. J Virol 2006;80:10208e17.60. Carr JM, Hocking H, Bunting K, Wright PJ, Davidson A,Gamble J, et al. Supernatants from dengue virus type-2infected macrophages induce permeability changes in endo-thelial cell monolayers. J Med Virol 2003;69:521e8.61. Talavera D, Castillo AM, Dominguez MC, Gutierrez AE, Meza I.IL8 release, tight junction and cytoskeleton dynamic reorga-nization conducive to permeability increase are induced bydengue virus infection of microvascular endothelial mono-layers. J Gen Virol 2004;85:1801e13.Autoimmunity in dengue 9
62. Chuang YC, Lei HY, Liu HS, Lin YS, Fu TF, Yeh TM. Macrophagemigration inhibitory factor induced by dengue virus infectionincreases vascular permeability. Cytokine 2011;54:222e31.63. Luplertlop N, Misse D, Bray D, Deleuze V, Gonzalez JP,Leardkamolkarn V, et al. Dengue-virus-infected dendriticcells trigger vascular leakage through metalloproteinaseoverproduction. EMBO Rep 2006;7:1176e81.64. Luplertlop N, Misse D. MMP cellular responses to dengue virusinfection-induced vascular leakage. Jpn J Infect Dis 2008;61:298e301.65. Suharti C, van Gorp EC, Setiati TE, Dolmans WM,Djokomoeljanto RJ, Hack CE, et al. The role of cytokines inactivation of coagulation and ﬁbrinolysis in dengue shocksyndrome. Thromb Haemost 2002;87:42e6.66. Rachman A, Rinaldi I. Coagulopathy in dengue infection andthe role of interleukin-6. Acta Med Indones 2006;38:105e8.67. Huerta-Zepeda A, Cabello-Gutierrez C, Cime-Castillo J, Mon-roy-Martinez V, Manjarrez-Zavala ME, Gutierrez-Rodriguez M,et al. Crosstalk between coagulation and inﬂammation duringdengue virus infection. Thromb Haemost 2008;99:936e43.68. Juffrie M, van Der Meer GM, Hack CE, Haasnoot K, Sutaryo,Veerman AJ, et al. Inﬂammatory mediators in dengue virusinfection in children: interleukin-8 and its relationship toneutrophil degranulation. Infect Immun 2000;68:702e7.69. Azeredo EL, Zagne SM, Santiago MA, Gouvea AS, Santana AA,Neves-Souza PC, et al. Characterisation of lymphocyteresponse and cytokine patterns in patients with dengue fever.Immunobiology 2001;204:494e507.70. Avirutnan P, Punyadee N, Noisakran S, Komoltri C,Thiemmeca S, Auethavornanan K, et al. Vascular leakage insevere dengue virus infections: a potential role for thenonstructural viral protein NS1 and complement. J Infect Dis2006;193:1078e88.71. Nascimento EJ, Silva AM, Cordeiro MT, Brito CA, Gil LH, Braga-Neto U, et al. Alternative complement pathway deregulationis correlated with dengue severity. PLoS One 2009;4:e6782.http://dx.doi.org/10.1371/journal.pone.0006782.72. Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M.Dengue virus infection of human endothelial cells leads tochemokine production, complement activation, andapoptosis. J Immunol 1998;161:6338e46.73. Lin YS, Yeh TM, Lin CF, Wan SW, Chuang YC, Hsu TK, et al.Molecularmimicrybetween virusandhostanditsimplicationsfordengue disease pathogenesis. Exp Biol Med 2011;236:515e23.74. Lin CF, Lei HY, Liu CC, Liu HS, Yeh TM, Wang ST, et al.Generation of IgM anti-platelet autoantibody in denguepatients. J Med Virol 2001;63:143e9.75. Oishi K, Inoue S, Cinco MT, Dimaano EM, Alera MT, Alfon JA,et al. Correlation between increased platelet-associated IgGand thrombocytopenia in secondary dengue virus infections. JMed Virol 2003;71:259e64.76. Saito M, Oishi K, Inoue S, Dimaano EM, Alera MT, Alfon JA,et al. Association of increased platelet-associated immuno-globulins with thrombocytopenia and the severity of diseasein secondary dengue virus infections. Clin Exp Immunol 2004;138:299e303.77. Falconar AK. The dengue virus nonstructural-1 protein (NS1)generates antibodies to common epitopes on human bloodclotting, integrin/adhesin proteins and binds to humanendothelial cells: potential implications in haemorrhagicfever pathogenesis. Arch Virol 1997;142:897e916.78. Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS, Yeh TM, et al. Anti-bodies from dengue patient sera cross-react with endothelialcells and induce damage. J Med Virol 2003;69:82e90.79. Markoff LJ, Innis BL, Houghten R, Henchal LS. Development ofcross-reactive antibodies to plasminogen during the immuneresponse to dengue virus infection. J Infect Dis 1991;164:294e301.80. Chungue E, Poli L, Roche C, Gestas P, Glaziou P, Markoff LJ.Correlation between detection of plasminogen cross-reactiveantibodies and hemorrhage in dengue virus infection. J InfectDis 1994;170:1304e7.81. Falconar AK. Antibody responses are generated to immuno-dominant ELK/KLE-type motifs on the nonstructural-1 glyco-protein during live dengue virus infections in mice andhumans: implications for diagnosis, pathogenesis, and vaccinedesign. Clin Vaccine Immunol 2007;14:493e504.82. Liu IJ, Chiu CY, Chen YC, Wu HC. Molecular mimicry of humanendothelial cell antigen by autoantibodies to nonstructuralprotein 1 of dengue virus. J Biol Chem 2011;286:9726e36.83. Lin CF, Lei HY, Lin YS, Liu CC, Anderson R. Patient and mouseantibodies against Dengue virus nonstructural protein 1 cross-react with platelets and cause their dysfunction or depletion.Am J Infect Dis 2008;4:69e75.84. Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM, Chen SH, et al.Endothelial cell apoptosis induced by antibodies againstdengue virus nonstructural protein 1 via production of nitricoxide. J Immunol 2002;169:657e64.85. Lin CF, Chiu SC, Hsiao YL, Wan SW, Lei HY, Shiau AL, et al.Expression of cytokine, chemokine, and adhesion moleculesduring endothelial cell activation induced by antibodiesagainst dengue virus nonstructural protein 1. J Immunol 2005;174:395e403.86. Lin CF, Wan SW, Chen MC, Lin SC, Cheng CC, Chiu SC, et al.Liver injury caused by antibodies against dengue virusnonstructural protein 1 in a murine model. Lab Invest 2008;88:1079e89.87. Cheng HJ, Lin CF, Lei HY, Liu HS, Yeh TM, Luo YH, et al.Proteomic analysis of endothelial cell autoantigens recog-nized by anti-dengue virus nonstructural protein 1 antibodies.Exp Biol Med 2009;234:63e73.88. Cheng HJ, Lei HY, Lin CF, Luo YH, Wan SW, Liu HS, et al. Anti-dengue virus nonstructural protein 1 antibodies recognizeprotein disulﬁde isomerase on platelets and inhibit plateletaggregation. Mol Immunol 2009;47:398e406.89. Chen MC, Lin CF, Lei HY, Lin SC, Liu HS, Yeh TM, et al. Deletionof the C-terminal region of dengue virus nonstructural protein1 (NS1) abolishes anti-NS1-mediated platelet dysfunction andbleeding tendency. J Immunol 2009;183:1797e803.90. Wan SW, Lin CF, Chen MC, Lei HY, Liu HS, Yeh TM, et al. C-terminal region of dengue virus nonstructural protein 1 isinvolved in endothelial cell cross-reactivity via molecularmimicry. Am J Infect Dis 2008;4:85e91.91. Chang HH, Shyu HF, Wang YM, Sun DS, Shyu RH, Tang SS, et al.Facilitation of cell adhesion by immobilized dengue viralnonstructural protein 1 (NS1): arginineeglycineeaspartic acidstructural mimicry within the dengue viral NS1 antigen. JInfect Dis 2002;186:743e51.92. Huang YH, Liu CC, Wang ST, Lei HY, Liu HL, Lin YS, et al.Activation of coagulation and ﬁbrinolysis during dengue virusinfection. J Med Virol 2001;63:247e51.93. Huang YH, Chang BI, Lei HY, Liu HS, Liu CC, Wu HL, et al.Antibodies against dengue virus E protein peptide bind tohuman plasminogen and inhibit plasmin activity. Clin ExpImmunol 1997;110:35e40.94. Chuang YC, Lei HY, Lin YS, Liu HS, Wu HL, Yeh TM. Denguevirus-induced autoantibodies bind to plasminogen andenhance its activation. J Immunol 2011;187:6483e90.95. Jardim DL, Tsukumo DM, Angerami RN, Carvalho Filho MA,Saad MJ. Autoimmune features caused by dengue fever:a case report. Braz J Infect Dis 2012;16:92e5.96. Rajadhyaksha A, Mehra S. Dengue fever evolving into systemiclupus erythematosus and lupus nephritis: a case report. Lupus2012;21:999e1002.97. Garcia G, Gonzalez N, Perez AB, Sierra B, Aguirre E, Rizo D,et al. Long-term persistence of clinical symptoms in dengue-10 S.-W. Wan et al.
infected persons and its association with immunologicaldisorders. Int J Infect Dis 2010;15:e38e43.98. Thomas SJ, Endy TP. Critical issues in dengue vaccine devel-opment. Curr Opin Infect Dis 2011;24:442e50.99. Schlesinger JJ, Brandriss MW, Walsh EE. Protection of miceagainst dengue 2 virus encephalitis by immunization with thedengue 2 virus non-structural glycoprotein NS1. J Gen Virol1987;68(Pt 3):853e7.100. Henchal EA, Henchal LS, Schlesinger JJ. Synergistic interac-tions of anti-NS1 monoclonal antibodies protect passivelyimmunized mice from lethal challenge with dengue 2 virus. JGen Virol 1988;69(Pt 8):2101e7.Autoimmunity in dengue 11