Characterization of early host responses in adults with dengue disease
© Tolfvenstam et al; licensee BioMed Central Ltd. 2011
Received: 24 January 2011
Accepted: 2 August 2011
Published: 2 August 2011
While dengue-elicited early and transient host responses preceding defervescence could shape the disease outcome and reveal mechanisms of the disease pathogenesis, assessment of these responses are difficult as patients rarely seek healthcare during the first days of benign fever and thus data are lacking.
In this study, focusing on early recruitment, we performed whole-blood transcriptional profiling on denguevirus PCR positive patients sampled within 72 h of self-reported fever presentation (average 43 h, SD 18.6 h) and compared the signatures with autologous samples drawn at defervescence and convalescence and to control patients with fever of other etiology.
In the early dengue fever phase, a strong activation of the innate immune response related genes were seen that was absent at defervescence (4-7 days after fever debut), while at this second sampling genes related to biosynthesis and metabolism dominated. Transcripts relating to the adaptive immune response were over-expressed in the second sampling point with sustained activation at the third sampling. On an individual gene level, significant enrichment of transcripts early in dengue disease were chemokines CCL2 (MCP-1), CCL8 (MCP-2), CXCL10 (IP-10) and CCL3 (MIP-1α), antimicrobial peptide β-defensin 1 (DEFB1), desmosome/intermediate junction component plakoglobin (JUP) and a microRNA which may negatively regulate pro-inflammatory cytokines in dengue infected peripheral blood cells, mIR-147 (NMES1).
These data show that the early response in patients mimics those previously described in vitro, where early assessment of transcriptional responses has been easily obtained. Several of the early transcripts identified may be affected by or mediate the pathogenesis and deserve further assessment at this timepoint in correlation to severe disease.
Dengue virus (DENV) is endemic throughout the tropics and in many subtropical parts of the world, causing significant human morbidity and mortality . An understanding of the host response, as obtained from genome-wide transcriptional profiling of dengue infection may reveal unique patterns pertaining to specific disease outcomes and identify molecular mechanisms that could be targeted pharmacologically [2, 3] and a number of studies of the host transcriptional responses to DENV infection have been performed. Comparing the results from in-vitro DENV-infected cells; HUVEC , non-small lung cancer cells , HepG2 [6, 7]; primary human immune cells  and muscle satellite cells ; to whole blood or isolated mononuclear cells from DENV-infected patients [10–14], the first studies reports abundant transcripts related to innate immunity while the latter largely report non-immune related transcripts of ER-stress, oxidative metabolism and signal transduction. However, the human studies sampled patients at the time of hospital admission or after 4-5 days of illness, focusing on differences between clinical phenotypes. We hypothesized that earlier assessment following symptom presentation would be required to characterize the in-vivo dengue innate immune response and that the early host responses may reflect components of the disease pathogenesis.
Characteristics of patients sampled for whole-blood RNA
DENV RT-PCR positive
DENV RT-PCR negative
Duration of fever to 1st sampling, hours
Male, no. (%)
DENV IgG positive at inclusion, no. (%)
Duration of symptoms to recovery, days
Sequential hematological parameters of DENV RT-PCR positive patients
Time from last sampling, hours
White blood cell count, 109/L
Thrombocyte count, 109/L
Differential expression measured by taq-man low density array in DENV RT-PCR positive patients
Acute dengue relative to convalescent dengue
Dengue at defervescence relative to convalescent dengue
Chemokine concentrations in consecutive serum samples from DENV RT-PCR positive patients
Median concentration in serum (pg/mL)
Acute dengue disease
Dengue at defervescence
p-value* acute vs convalescence
Taken together, at this previously unassessed early stage of dengue disease the innate immune responses predominate, with the most significant canonical pathways being IFN-signaling, pattern recognition signaling and complement activation, both in relation to autologous transcripts in convalescence and to whole blood transcripts in non-dengue febrile illnesses at a similar phase following fever onset. In the convalescent phase of dengue disease, pathways related to adaptive immune responses are active, rendering these genes to appear down-regulated at the acute phase and in concordance with previous reports on transcripts derived from dengue disease in or just before defervescence [10–14], non-immune canonical pathways dominate. Interestingly, many of the responses seen in acute dengue in relation to the convalescent baseline were replicated when comparing to other non-dengue febrile illnesses, indicating a more prominent IFN-response, specific to dengue disease, and a selective utlization of TLR7, MDA5 and OAS. We do not have information of the etiology of the non-dengue febrile controls, but etiological search in patients enrolled later in the same cohort have shown that viral infections such as influenza, adenovirus and metapneumovirus are common among the DENV RT-PCR negative controls. IFN-responses arise hours after viremia is established and are likely of great importance for the control of viral replication. The IFN-response in dengue infection have been shown to be activated through two main pathogen recognition families; the TLRs and the RLRs. Of the TLRs, TLR7 in dendritic cells has been shown to interact with DENV RNA leading to viral fusion and uncoating processing that in the end activated a type-1 IFN-response . TLR3 was also shown to have a role in the regulation of the inflammatory response in dengue infected umbilical vein endothelial cells . When we compared the acute samples with the samples collected at convalescence in our dengue positive patients we saw an up regulation of TLR7 and IRF7 which indicates an activation of the TLR7 signalling pathway. TLR7 is an intracellular receptor that senses microbial nucleic acids and via IRF7 induces a strong type-1 IFN-response, particularly IFNα ; while it can also induced a type-1 IFN-response via NFκB . RLR activation was also seen when comparing acute versus convalecent samples in the DENV RT-PCR positive patients, with the genes RIG1 and MDA5 upregulated. A recent study from Fredericksen et al demonstrated that RIG1 and MDA5 induce an IFN-response in West Nile virus-infected fibroblasts by activation of IRF3 . They also show that RIG1 primes an early IFN-response while MDA5 is more involved in the second phase of IFN-dependent gene expression. Both RIG1 and MDA5 have also been shown to be activated in double RIG1/MDA5 knockout mouse fibroblasts [28, 29]. Interestingly, RIPK2 (RIP2) was also identified as an over expressed gene indicating activation of the NLR signalling pathway. NOD1 and NOD2, members of the NLR protein family, are activated by specific bacterial peptides, and via RIPK2 induce NFκB activation . There are no earlier reports on virus induced NLR activation. Transcripts clustering to the PPAR pathway were also among the most significantly enriched comparing dengue to non-dengue. PPARγ has been shown to play a critical role in the control of adipocyte differentiation and lipid metabolism, and also immunity and the barrier functions of epithelial and endothelial cells; in dengue disease this could be a response to epithelial stress [31, 32]. Furthermore, among the most highly enriched gene transcripts is JUP (plakoglobin), that encode a protein that forms part of desmosomes and intermediate junctions in endothelial cells, wich could indicate that some transcripts were derived from affected endothelial cells. In fact, high numbers of detached endothelial cells in peripheral blood has been obseved in the acute phase of dengue . DEFB1 was another highly enriched transcript which encodes an antimicrobial peptide, β- defensin 1, that has mostly been studied in the context of epithelial protection against HIV . Also among the highly enriched transcripts were genes of the secreted mediators CCL2, CCL8, CXCL10 and CCL3, whose proteins were also found to be adundant in patient serum (Table 2). These have been observed before in dengue disease and MCP-1 could well participate in the pathogenesis of vascular leakage by its effect on endothelial tight junctions [35, 36]. Other genes that were significantly enriched but did not cluster to any canonical pathway were NMES1 and CCNA1. While CCNA1 is a cyclin which probably mediates cell cycle arrest to prevent virus replication in infected cells, little is known about the gene NMES1. A recent study has identified that the transcript is a primary functional microRNA (miR-147) which was expressed in murine macrophages upon TLR-stimulation . It was shown to negatively regulate inflammatory cytokine expression in these cells. As dengue virus is believed to primarily replicate in cells of monocyte/macrophage lineage, NMES1 expression may be important to moderate the pro-inflammatory cytokine release from these cells, especially as these are potential mediators of pathology. Comparing dengue patients with different DENV-serostatus at inclusion rendered a surprisingly small number of differentially expressed genes with no significant pathway clustering. On the individual gene level PRDX2, an antioxidant enzyme was enriched in the acute stage of secondary DENV infection, while at defervesence, CCR2 was among the transcripts seen upregulated in secondary infection. Sierra et al. addressed this issue by examining in-vitro infected PBMC from immune and non-immune individuals and showed expression of CCL2 to be highly dependent on previously infecting serotype at 24 h post infection . CCL2 was not an found an enriched transcript in acute secondary infection in our material but the number of patients in each group in this specific comparison was small (ten patients were DENV RT-PCR and DENV-IgG positive) and information of previous infecting serotype was lacking, thus this specific assessment should be perfomed in a larger cohort to allow further speculation on these genes in the context of dengue pathogenesis.
In summary, early DENV-induced transcriptional host responses in-vivo are predominatly involving innate immune responses and overlaps to a large extent with those described in in-vitro, where early transcriptional assessment post-infection has been easily obtained. Several of the early transcripts identified deserve further assessment in correlation to severe disease.
This work was funded in part by the Singapore Agency for Science, Technology and Research (A*STAR) and by the National Medical Research Council of Singapore, who had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Global burden of dengue. [http://www.pdvi.org]
- Jenner RG, Young RA: Insights into host responses against pathogens from transcriptional profiling. Nat Rev Microbiol. 2005, 3: 281-294. 10.1038/nrmicro1126.View ArticlePubMed
- Katze MG, Fornek JL, Palermo RE, Walters KA, Korth MJ: Innate immune modulation by RNA viruses: emerging insights from functional genomics. Nat Rev Immunol. 2008, 8: 644-654. 10.1038/nri2377.View ArticlePubMed
- Warke RV, Xhaja K, Martin KJ, Fournier MF, Shaw SK, Brizuela N, de Bosch N, Lapointe D, Ennis FA, Rothman AL, Bosch I: Dengue virus induces novel changes in gene expression of human umbilical vein endothelial cells. J Virol. 2003, 77: 11822-11832. 10.1128/JVI.77.21.11822-11832.2003.PubMed CentralView ArticlePubMed
- Nasirudeen AM, Liu DX: Gene expression profiling by microarray analysis reveals an important role for caspase-1 in dengue virus-induced p53-mediated apoptosis. J Med Virol. 2009, 81: 1069-1081. 10.1002/jmv.21486.View ArticlePubMed
- Conceicao TM, El-Bacha T, Villas-Boas CS, Coello G, Ramirez J, Montero-Lomeli M, Da Poian AT: Gene expression analysis during dengue virus infection in HepG2 cells reveals virus control of innate immune response. J Infect. 2009, 60: 65-75.View ArticlePubMed
- Fink J, Gu F, Ling L, Tolfvenstam T, Olfat F, Chin KC, Aw P, George J, Kuznetsov VA, Schreiber M, Vasudevan SG, Hibberd ML: Host gene expression profiling of dengue virus infection in cell lines and patients. PLoS Negl Trop Dis. 2007, 1: e86-10.1371/journal.pntd.0000086.PubMed CentralView ArticlePubMed
- Becerra A, Warke RV, Martin K, Xhaja K, de Bosch N, Rothman AL, Bosch I: Gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo. J Med Virol. 2009, 81: 1403-1411. 10.1002/jmv.21538.PubMed CentralView ArticlePubMed
- Warke RV, Becerra A, Zawadzka A, Schmidt DJ, Martin KJ, Giaya K, Dinsmore JH, Woda M, Hendricks G, Levine T, Rothman AL, Bosch I: Efficient dengue virus (DENV) infection of human muscle satellite cells upregulates type I interferon response genes and differentially modulates MHC I expression on bystander and DENV-infected cells. J Gen Virol. 2008, 89: 1605-1615. 10.1099/vir.0.2008/000968-0.View ArticlePubMed
- Long HT, Hibberd ML, Hien TT, Dung NM, Van Ngoc T, Farrar J, Wills B, Simmons CP: Patterns of gene transcript abundance in the blood of children with severe or uncomplicated dengue highlight differences in disease evolution and host response to dengue virus infection. J Infect Dis. 2009, 199: 537-546. 10.1086/596507.PubMed CentralView ArticlePubMed
- Nascimento EJ, Braga-Neto U, Calzavara-Silva CE, Gomes AL, Abath FG, Brito CA, Cordeiro MT, Silva AM, Magalhaes C, Andrade R, Gil LH, Marques ET: Gene expression profiling during early acute febrile stage of dengue infection can predict the disease outcome. PLoS One. 2009, 4: e7892-10.1371/journal.pone.0007892.PubMed CentralView ArticlePubMed
- Simmons CP, Popper S, Dolocek C, Chau TN, Griffiths M, Dung NT, Long TH, Hoang DM, Chau NV, Thao le TT, Hien TT, Relman DA, Farrar J: Patterns of host genome-wide gene transcript abundance in the peripheral blood of patients with acute dengue hemorrhagic fever. J Infect Dis. 2007, 195: 1097-1107. 10.1086/512162.PubMed CentralView ArticlePubMed
- Ubol S, Masrinoul P, Chaijaruwanich J, Kalayanarooj S, Charoensirisuthikul T, Kasisith J: Differences in global gene expression in peripheral blood mononuclear cells indicate a significant role of the innate responses in progression of dengue fever but not dengue hemorrhagic fever. J Infect Dis. 2008, 197: 1459-1467. 10.1086/587699.View ArticlePubMed
- Loke P, Hammond SN, Leung JM, Kim CC, Batra S, Rocha C, Balmaseda A, Harris E: Gene expression patterns of dengue virus-infected children from nicaragua reveal a distinct signature of increased metabolism. PLoS Negl Trop Dis. 2010, 4: e710-10.1371/journal.pntd.0000710.PubMed CentralView ArticlePubMed
- Low JG, Ooi EE, Tolfvenstam T, Leo YS, Hibberd ML, Ng LC, Lai YL, Yap GS, Li CS, Vasudevan SG, Ong A: Early Dengue infection and outcome study (EDEN) - study design and preliminary findings. Ann Acad Med Singapore. 2006, 35: 783-789.PubMed
- Dengue: guidelines for diagnosis, treatment, prevention and control -- New edition. [http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf]
- Lye DC, Chan M, Lee VJ, Leo YS: Do young adults with uncomplicated dengue fever need hospitalisation? A retrospective analysis of clinical and laboratory features. Singapore Med J. 2008, 49: 476-479.PubMed
- Ito M, Takasaki T, Yamada K, Nerome R, Tajima S, Kurane I: Development and evaluation of fluorogenic TaqMan reverse transcriptase PCR assays for detection of dengue virus types 1 to 4. J Clin Microbiol. 2004, 42: 5935-5937. 10.1128/JCM.42.12.5935-5937.2004.PubMed CentralView ArticlePubMed
- Schreiber MJ, Holmes EC, Ong SH, Soh HS, Liu W, Tanner L, Aw PP, Tan HC, Ng LC, Leo YS, Low JG, Ong A, Ooi EE, Vasudevan SG, Hibberd ML: Genomic epidemiology of a dengue virus epidemic in urban Singapore. J Virol. 2009, 83: 4163-4173. 10.1128/JVI.02445-08.PubMed CentralView ArticlePubMed
- Hoang LT, Lynn DJ, Henn M, Birren BW, Lennon NJ, Le PT, Duong KT, Nguyen TT, Mai LN, Farrar JJ, Hibberd ML, Simmons CP: The early whole-blood transcriptional signature of dengue virus and features associated with progression to dengue shock syndrome in Vietnamese children and young adults. J Virol. 2010, 84: 12982-12994. 10.1128/JVI.01224-10.PubMed CentralView ArticlePubMed
- Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001, 98: 5116-5121. 10.1073/pnas.091062498.PubMed CentralView ArticlePubMed
- Edgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002, 30: 207-210. 10.1093/nar/30.1.207.PubMed CentralView ArticlePubMed
- Burgner D, Davila S, Breunis WB, Ng SB, Li Y, Bonnard C, Ling L, Wright VJ, Thalamuthu A, Odam M, Shimizu C, Burns JC, Levin M, Kuijpers TW, Hibberd ML: A genome-wide association study identifies novel and functionally related susceptibility Loci for Kawasaki disease. PLoS Genet. 2009, 5: e1000319-10.1371/journal.pgen.1000319.PubMed CentralView ArticlePubMed
- Wang JP, Liu P, Latz E, Golenbock DT, Finberg RW, Libraty DH: Flavivirus activation of plasmacytoid dendritic cells delineates key elements of TLR7 signaling beyond endosomal recognition. J Immunol. 2006, 177: 7114-7121.View ArticlePubMed
- Kumar H, Kawai T, Akira S: Toll-like receptors and innate immunity. Biochem Biophys Res Commun. 2009, 388: 621-625. 10.1016/j.bbrc.2009.08.062.View ArticlePubMed
- Diamond MS: Mechanisms of evasion of the type I interferon antiviral response by flaviviruses. J Interferon Cytokine Res. 2009, 29: 521-530. 10.1089/jir.2009.0069.View ArticlePubMed
- Fredericksen BL, Keller BC, Fornek J, Katze MG, Gale M: Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1. J Virol. 2008, 82: 609-616. 10.1128/JVI.01305-07.PubMed CentralView ArticlePubMed
- Chang TH, Liao CL, Lin YL: Flavivirus induces interferon-beta gene expression through a pathway involving RIG-I-dependent IRF-3 and PI3K-dependent NF-kappaB activation. Microbes Infect. 2006, 8: 157-171. 10.1016/j.micinf.2005.06.014.View ArticlePubMed
- Loo YM, Fornek J, Crochet N, Bajwa G, Perwitasari O, Martinez-Sobrido L, Akira S, Gill MA, Garcia-Sastre A, Katze MG, Gale M: Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J Virol. 2008, 82: 335-345. 10.1128/JVI.01080-07.PubMed CentralView ArticlePubMed
- Krieg A, Correa RG, Garrison JB, Le Negrate G, Welsh K, Huang Z, Knoefel WT, Reed JC: XIAP mediates NOD signaling via interaction with RIP2. Proc Natl Acad Sci USA. 2009, 106: 14524-14529. 10.1073/pnas.0907131106.PubMed CentralView ArticlePubMed
- Huang W, Eum SY, Andras IE, Hennig B, Toborek M: PPARalpha and PPARgamma attenuate HIV-induced dysregulation of tight junction proteins by modulations of matrix metalloproteinase and proteasome activities. FASEB J. 2009, 23: 1596-1606. 10.1096/fj.08-121624.PubMed CentralView ArticlePubMed
- Ogasawara N, Kojima T, Go M, Ohkuni T, Koizumi J, Kamekura R, Masaki T, Murata M, Tanaka S, Fuchimoto J, Himi T, Sawada N: PPARgamma agonists upregulate the barrier function of tight junctions via a PKC pathway in human nasal epithelial cells. Pharmacol Res. 2010, 61: 489-498. 10.1016/j.phrs.2010.03.002.View ArticlePubMed
- Cardier JE, Rivas B, Romano E, Rothman AL, Perez-Perez C, Ochoa M, Caceres AM, Cardier M, Guevara N, Giovannetti R: Evidence of vascular damage in dengue disease: demonstration of high levels of soluble cell adhesion molecules and circulating endothelial cells. Endothelium. 2006, 13: 335-340. 10.1080/10623320600972135.View ArticlePubMed
- Prado-Montes de Oca E: Human beta-defensin 1: a restless warrior against allergies, infections and cancer. Int J Biochem Cell Biol. 2010, 42: 800-804. 10.1016/j.biocel.2010.01.021.View ArticlePubMed
- Lee YR, Liu MT, Lei HY, Liu CC, Wu JM, Tung YC, Lin YS, Yeh TM, Chen SH, Liu HS: MCP-1, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells. J Gen Virol. 2006, 87: 3623-3630. 10.1099/vir.0.82093-0.View ArticlePubMed
- Stamatovic SM, Keep RF, Kunkel SL, Andjelkovic AV: Potential role of MCP-1 in endothelial cell tight junction 'opening': signaling via Rho and Rho kinase. J Cell Sci. 2003, 116: 4615-4628. 10.1242/jcs.00755.View ArticlePubMed
- Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E: miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Natl Acad Sci USA. 2009, 106: 15819-15824. 10.1073/pnas.0901216106.PubMed CentralView ArticlePubMed
- Sierra B, Perez AB, Vogt K, Garcia G, Schmolke K, Aguirre E, Alvarez M, Volk HD, Guzman MG: MCP-1 and MIP-1alpha expression in a model resembling early immune response to dengue. Cytokine. 2010, 52: 175-183. 10.1016/j.cyto.2010.06.010.View ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/209/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.