DENGUE HAEMORRHAGIC FEVERGINO TANN

Wilder-Smith, A. et al. N Engl J Med 2005;353:924-932World Distribution of Dengue, 200250100 million cases of DF 250,000500,000 cases of DHF occur every year

Dengue Virus Single-stranded RNA virusCauses dengue hemorrhagic fever Transmitted by mosquitoes (Aedes aegypti)Serotypes (DEN-1, 2, 3, 4)

Dengue VirusNature Structural Biology 10, 907 - 912 (2003)

The dengue viral genome

Copyright 2004 American Society for Clinical InvestigationRothman, A. L. J. Clin. Invest. 2004;113:946-951Organization of the flavivirus genome and its resulting proteins and the location of the major targets of the immune response

Life Cycle of Dengue VirusNature Reviews Microbiology 3, 13-22 (January 2005) Structural changes in envelope protein, icosahedral shell:Fusion antiparallel homodimers to parallel homotrimersMaturation conformational change of immature trimers to homodimers followed by protease cleavage (by furin) to form mature antiparpllel dimers

Intracellular life cycle of dengue virus Journal of Virology, December 2006, p. 11418-11431, Vol. 80, No. 23

Maturation of Dengue VirusScience 28 March 2008:Vol. 319. no. 5871, pp. 1834 - 1837

Analysis of CD14 anti-actin anti-HSP70 anti-HSP90 control antibody HSP90 and HSP70 on the surface of peripheral blood monocytes Journal of Virology, April 2005, p. 4557-4567, Vol. 79, No. 8

Dose and time dependence of DV infection in THP DC-SIGN cellsTassaneetrithep et al. Journal of Experimental Medicine 2008:197:823-829 2008 Tassaneetrithep et.al.DC-specific intracellular adhesion molecule (ICAM) 3grabbing nonintegrin (DC-SIGN)

DEN-2 antigen-positive ECV304 cells Endothelial cell line ECV304, derived from human umbilical cord J Gen Virol 84 (2003), 3095-3098

Infection inhibition assays by anti-HSP90 and anti-HSP70

class II viral envelope proteins require acidic pH in different intracellular compartments trigger viral fusion with the host membrane clathrin-dependent endocytosis colocalize with both early and late endosomes requires microtubules vacuolar ATPase (VATPase) endosomotropic weak base NH4Cl small interfering RNAs (siRNAs) targeting VATPase receptor-mediated endocytosis inhibitor chlorpromazine Rab 5 and 7 GTPases regulators of transport to early and late endosomes

Eps15, a gene required for clathrin-dependent endocytosis HeLa cells

Dominant-negative mutants of Rab 5 (Rab 5 S34N) and 7 (Rab 7 T22N)

Innate immune encounter upon virus injection in the capillary vessel Experimental Biology and Medicine 233:401-408 (2008)

Pathogenesis of dengue virus infection Journal of Virology, December 2006, p. 11418-11431, Vol. 80, No. 23

VIRAL RISK FACTORS FOR DHF PATHOGENESIS

Virus strain (genotype) Epidemic potential: viremia level, infectivity Virus serotype

DHF risk is greatest for DEN-2, followed by DEN-3, DEN-4 and DEN-1 Present pandemic, subtype 3, from India

CLINICAL CASE DEFINITION FOR DHF

4 Necessary Criteria:Fever, or recent history of acute fever Haemorrhagic manifestations Low platelet count (100,000/mm3 or less) Objective evidence of leaky capillaries: elevated haematocrit (20% or more over baseline) low albumin pleural or other effusions

Four clinical scenarios of increasing severity

DISEASE COURSE

WARNING SIGNS

Wilder-Smith, A. et al. N Engl J Med 2005;353:924-932Dengue Rash with Sparing of Islands of Skin

Wilder-Smith, A. et al. N Engl J Med 2005;353:924-932Positive Result on a Tourniquet Test

DENGUE HAEMORRHAGIC FEVER

DENGUE ENCEPHALITIS

RIGHT LATERAL DECUBITUS POSITION

Box 1: Differential diagnosis of dengue fever and DHF Dengue fever

Infectious mononucleosis. Chickengunya viral infections. Coxsackie and other enteroviral infections. Rickettsial infections. Rubella. Parvovirus B19 infections. Leptospirosis. Influenza. DHF Leptospirosis. Chikengunya viral infections. Kawasaki disease. Yellow fever. Hanta viral infections. Other viral haemorrhagic fevers. Meningococcal septicaemia.

DIAGNOSISFeverRetro-orbital pain, photophobiaMyalgia, arthralgiaFlushingTorniquet testThrombocytopenis < 100 000Leukopenia < 4 000Dengue SerologyIf negative maybe not dengue - too early, repeat in 3-5 days - Chikungunya - Chlamydia, test for IgM Chlamydia

If positive - IgM (+), IgG (+) = secondary infection - IgM (+), IgG (-) = primary infection - IgM (-), IgG (+) = maybe secondary infection maybe previous infection

Wilder-Smith, A. et al. N Engl J Med 2005;353:924-932Laboratory Diagnosis of Dengue

PROBABLE DHF DIAGNOSEDAvoid salicylates (Chinese medicine, minyak angin)

Avoid NSAIDs (Can cause Reyes syndrome)

Avoid vitamin C and E (acidic fruits, jambu)

DO NOT massage or coin (kerok) the patient

Hospitalize all with platelets < 100 000

PARACETAMOL< 1 year 60 mg/dose1-3 years 60-120 mg/dose3-6 years 120 mg/dose6-12 years 240 mg/dose

No more than 6 doses in 24 hours

HAEMODYNAMIC IMPAIRMENTMild pulse pressure > 20 mm Hg

Moderate 10 20 mm Hg

Severe < 10 mm HgMajor pathologic problem acute loss of plasma in the vascular compartment, up to 20 % in severe cases.

Shock, tissue anoxia, metabolic acidosis, death.

FLUID MANAGEMENT IN CHILDREN 1st hour 15 ml/kg bw 2nd hour 10 ml/kg bwMonitor pulse, BP and perfusion hourly until stablefor at least 24 hours

Reduce fluid to 5 ml/kg, 3 mg/kg etc (refer to maintenance fluid requirements

Monitor haematocrit at 0, 2, 6, 12 and every 12 hours

Coagulation screen at day 2 and 4

Ultrasound scan of chest and abdomenIf pulse pressure < 20, rising haematocrit, rescue with 5-10 ml/kg fluid

MAINTENANCE FLUIDBody weight (kg) Volume over 24 hours

100/kg10-20 1000 + 50 for each kg in excess of 10>20 1500 + 20 for each kg in excess of 20

Halliday MA, Segar WE. Pediatrics 1957; 19:82310 ml/kg bw for each 1% of body weight lost plusmaintenance fluid

Ringers solution 2 liters a day

> 2 liters, > pulmonary edemaFLUID IN ADULTS

Wills, B. A. et al. N Engl J Med 2005;353:877-889Kaplan-Meier Curves for Time from Study Entry to Initial (Panel A) and Sustained (Panel B) Cardiovascular Stability among Children in Group 1, According to the Resuscitation Fluid Received

SIGNS OF FLUID RETENSION PULMONARY EDEMAFurosemide start with 2 mg/kg total dose given a a continuous infusion. DO NOT GIVE BOLUSES !Adjust dose as needed, depending on haemodynamics.

HAEMOSTATIC CHANGESCapillary fragility (no vasculitis)

Thrombocytopenia

Disorders of coagulationMajor bleeding is mainly from the GI TractMajor Bleed give fresh frozen plasma 15 ml/kg - platelets preferably single donor

DO NOT TREAT THE PLATELET COUNT, TREAT THE PATIENT !!!

Significant Minor Bleed tranexamic acid 1 g IV, repeat another dose in one hour. Children, adjust dose accordingly.

antibody-dependent enhancement cell-mediated pathogenesis cytokine storm phenomenon genetic background virus strain differences levels of virus circulating in individuals nutritional status Hypotheses on the Pathogenesis of DHF/DSS

hyper-thermal factors physical status of virus in viremic individuals conditioning of neutralizing antibody assay concept of vector transmission innate immune system Alternate scenarios

fever inducers TNF-alpha, IL-1 and IL-6, fever inhibitors TGF-beta and IL-10 Thermal response following dengue virus infection Hyper-thermal conditions have been reported to increase cell susceptibility to virus infection

virus in plasma (free viral particle) Viremia cell associated platelets, lymphocytes, monocytes cell-associated viruses are poorly cleared plasma associated virus is 95% cleared in less than 2 mins cell-associated virus helps spread the virus throughout the body circulate in the form of immune-complexes

Copyright 2004 American Society for Clinical InvestigationRothman, A. L. J. Clin. Invest. 2004;113:946-951Proposed model of heterologous immunity in secondary dengue virus infections and its implications for the pathogenesis of dengue hemorrhagic feverFlaviviruses share peptide sequences

Copyright 2004 American Society for Clinical InvestigationRothman, A. L. J. Clin. Invest. 2004;113:946-951Postulated pathologic mechanisms of DENV-reactive immune responses in the development of DHFantibody-dependent enhancement

ANTI-PLATELET ANTIBODIES IN DENGUE-2 INFECTION IN MICEHuang KJ et al. Journal of General Virology (2000), 81, 2177-2182A role for antibody mimicry

Thrombocytopenia Destruction of platelets by the virus itself (direct cytotoxicity) Immune-mediated toxicity dengue virus RNA purified from platelets from dengue infected patients reservoir for dengue virus replication platelet transfusions add fuel to fire ?

Steroid Therapy Has No Additional Benefit in Dengue Thrombocytopenia.N Alam, M A Hasanat, M Moslehuddin-Ahmed, M M Aziz.Samorita Hospital, Dhaka, Bangladesh; Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh; Immunology, BIRDEM, Dhaka, Bangladesh.CONCLUSIONS: This study demonstrated that dexamethasone therapy for thrombocytopenia has no beneficial effect in dengue fever. It may rather hinder the natural recovery process of platelet increment in dengue viral infection. ICI/FOCIS Abstract 1227, 2004

Copyright 2004 American Society for Clinical InvestigationRothman, A. L. J. Clin. Invest. 2004;113:946-951Immunological considerations and current status in development of candidate dengue vaccines

Monath T. N Engl J Med 2007;357:2222-2225Vaccines against Dengue and Yellow Fever

A Lesson from Latin America

AEDES AEGYPTIFemale feeds on bloodDaytimeLife-span 2-3 monthsLays 100 eggs / 2 daysCan fly 100 meters

LIFE CYCLELARVA lives in clean water. Can also Thrive in dirty water.

Water container in and around the house7 10 days adult mosquito

Preillness gaps biologic transmission mechanical transmission

Role of innate immune parameters in DHF/DSS CD5+ B cells (B1 cells)Natural IgM antibodies Pentameric IgM Hexameric IgM, IgG, and IgA natural antibodies Platelet-associated IgM

Development of an image-based immunofluorescence assay for detection of DENVChu J. J. H., Yang P. L. PNAS 2007;104:3520-35252007 by National Academy of Sciences

Antiviral activity of dasatinib, an inhibitor of c-Src and Abl kinases, on DENV infectionChu J. J. H., Yang P. L. PNAS 2007;104:3520-35252007 by National Academy of Sciences

Inhibitors of cyclin-dependent kinases (K002), c-Raf (K039), the JAK (K040), and the multitargeted inhibitor imatinib (K014), seemed to affect events early in viral infection (e.g., viral entry, genome release) because incubation of host cells with thesecompounds for a three-hour period before inoculation was sufficient to cause significant reductions in DENV titer Five compounds collectively targeting Src-family (K003, K030,K117), Abl (K003, K013), c-Kit (K003), PDGFR (K003), VEGFR (K003, K030) kinases and casein kinase II (CK II, K032) were found to have anti-DENV activity in both pre- and postinoculation assays. These data suggested that these compounds may inhibit kinases that are important for multiple viral processes that occur during or after viral entry.

Inhibition of viral spread in dasatinib-treated, DENV-infected cellsChu J. J. H., Yang P. L. PNAS 2007;104:3520-35252007 by National Academy of Sciences

siRNA Knockdown of c-Src protein levels inhibits DENV infectionChu J. J. H., Yang P. L. PNAS 2007;104:3520-35252007 by National Academy of Sciences

Inhibition of c-Src protein kinase activity prevents the assembly of DENV within the ER lumenChu J. J. H., Yang P. L. PNAS 2007;104:3520-35252007 by National Academy of Sciences

THANK YOUGT26112005GT01122009

Figure 1. World Distribution of Dengue, 2002. Adapted from the Centers for Disease Control and Prevention.8About dengue virus and how it infects humans to cause dengue fever.Elelctron micrograph showing the structure of dengue virusThe dengue viral genome. Noncoding regions with their terminal structures are indicated by black lines. The single open reading frame encodes a poly-protein that is processed by the viral NS2B-NS3 protease and cell proteases to the mature viral proteins. Structural proteins are C, prM, and E. Nonstructural proteins are 1, 2A, 2B, 3, 4A, 4B, and 5. The genome is not drawn to scale Structure of the dengue virus genome. DENV vRNA contains a 5' type 1 cap structure, and the single open reading frame is flanked by 5' and 3' UTRs. Conserved RNA secondary structures in the UTRs and in the coding region have been determined to function at various stages of the viral life cycle. Functions for several of the viral proteins have been determined; however, many remain to be elucidated. The secondary structure is based on Southeast Asian strains of DENV2 and is similar for different strains and serotypes. Structures designated cHP, CS, UAR, DB1, DB2 and 3'SL were compiled from a combination of computer predictions, functional analyses, and solution structure probing (3, 34, 71, 132, 148, 154). RdRP, RNA-dependent RNA polymerase; DB, dumbbe The life cycle of dengue virus from the point that it infects a host cell to the release of new mature virusIntracellular life cycle of dengue virus. DENV binds (step 1) and enters (step 2) cells via an uncharacterized receptor(s) by RME. Endosomal acidification (step 3) results in an irreversible trimerization of the viral E protein, exposing the fusion domain. After being uncoated, the vRNA is translated (step 4) at ER-derived membranes, where it is processed into three structural and seven NS proteins. After the viral replication complex is synthesized, vRNA translation switches off and RNA synthesis (step 5) begins. Subsequently, successive rounds of translation (step 6) are followed by assembly in the ER. The virion is maturated in the Golgi compartment (step 7) and exits via the host secretory pathway. Viral proteins C, E, NS3, and NS5 have been observed in the nuclei of infected cells (57, 96, 177, 186, 190), and the vRNA associates specifically with a number of cellular proteins (41, 58, 187, 198); however, the biological significance is unknown. L-SIGN, liver/lymph node-specific ICAM-3-grabbing nonintegrin; PTB, polypyrimidine tract binding protein; EF-1 , elongation factor 1 ; hnRNP L, heterogeneous nuclear ribonucleoprotein L; PDI, protein disulfide isomerase Maturation of the envelope protein: the knob like structures in the immature protein (colored blue in the figure), are cleaved by a host enzyme resulting in relatively smooth mature virus, that can now infect another cell. Localization of HSP90 and HSP70 on the surface of peripheral blood monocytes. (A) Analysis of CD14 surface expression in 10-day-old peripheral blood monocytes/macrophages (solid line) and the control antibody of isotype IgG (dashed line) by flow cytometry. (B) Nonpermeabilized 10-day-old monocytes/macrophages were incubated with anti-actin (b), anti-HSP70 (d), and anti-HSP90 (e), or a control antibody (c), followed by staining with a fluorescein isothiocyanate-conjugated goat anti-mouse IgG (a to e). A phase contrast image is shown in panel a. Dose and time dependence of DV infection in THP DC-SIGN cells. (A) Titration of DV2 infection in THP DC-SIGN and THP-1 2 d after infection. The infected cells were stained for DV envelope antigen (clone 2H2) and the NS1 (clone 7E11) and infection was determined by calculating the percentage of fluorescence-positive cells. (B) Kinetics of DV2 infection in THP DC-SIGN and THP-1. The infected cells were harvested at t = 0, 24, 48, and 72 h and stained with 2H2 and 7E11. (C) DV infection rates of all four DV serotypes in THP DC-SIGN and THP-1, using DV 1, 2, 3, and 4. Data are averages of two independent experiments. (D) Representative immunofluorescence experiment at two time points (2 h, top, and 24 h, bottom) showing bound DV envelope complex antigens (red) in DC-SIGN (green)bearing DC (left) and THP-DC-SIGN (right) cells. Nuclei were labeled with DAPI stain. (E) Representative immunohistochemistry experiment showing bound intracellular DV envelope complex antigens (dark red) in DC-SIGNbearing cells (blue). The left (immature DC) and right panels (THP DC-SIGN) show cells infected with DV. Original magnifications at 200 (insets, 600).Percentage of DEN-2 antigen-positive ECV304 cells collected at different times p.i. at various m.o.i. (a) ECV304 cells pre-incubated with various concentrations of rEgp, infected with DEN-2 at an m.o.i. of 10 and collected at different times p.i. (b) Infected ECV304 cells were scraped and harvested. DEN-2 antigen-positive cells were counted by IFA Infection inhibition assays by anti-HSP90 and anti-HSP70 antibodies in peripheral blood monocytes/macrophages. Monocytes/macrophages were preincubated with different concentrations of an unrelated antibody (mouse polyclonal serum; diamond) or with anti-HSP90 (square) and anti-HSP70 (triangle) antibodies for 1 h at 37C. Subsequently, cells were infected with DEN-2 at an MOI of 5. Culture supernatants were harvested after 48 h of infection and assayed to determine the infectious virus titer. Each experimental point is presented as the mean of results obtained from three separate experiments Effect of NH4Cl (A) and chlorpromazine (D) and depletion of VATPase and Eps15 (B, C, E, and F) on DNV or WNV infectivity of HeLa cells. (A and D) HeLa cells preadsorbed with the viruses for 1 h at 4C (MOI of 10) were transferred to 37C and incubated for the desired time periods, culture medium containing 20 mM NH4Cl or 10 M chlorpromazine was added, and the cells were grown for 16 h or 10 h for DNV or WNV, respectively. After the incubation, cells were fixed and immunofluorescence was performed. (B and C) HeLa cells transfected with siRNA against VATPase and Eps15 for 4 days were infected with DNV and WNV (MOI of 10) for 16 h or 10 h, respectively, and analyzed by immunofluorescence (B) or Q-RT-PCR to quantify viral E-gene copies (C). (E and F) HeLa cells transfected with pEGFP-C vector expressing a dominant-negative mutant of Eps15 (E95/295) for 30 h were infected with DNV or WNV (MOI of 10) for 16 h or 10 h, respectively, and analyzed by immunofluorescence (E) or Q-RT-PCR (F) to quantify viral E-gene copies. E-gene copies were normalized using beta actin gene copies. Images were captured using a magnification x40 objective by fluorescence microscopy. Quantification was done by counting the total number of infected cells in each captured image (corresponds to an average of 100 cells per image) and expressed as percent infected cells of the total cells. Results are expressed as means standard deviations from triplicates of a representative experiment Virus-mediated, pH-induced cell-cell fusion by DNV and WNV. Cells (labeled with the fluorescent probe CellTracker Green CMFDA; Molecular Probes) infected with virus for 20 h (MOI of 10) or preadsorbed with virus on surface by incubation with virus at an MOI of 200 for 1 h at 4C (both labeled with the fluorescent probe CellTracker Green CMFDA) were mixed at a 1:1 ratio at 1 x 106 cells/ml in buffers of various pHs for up to 5 min at 37C. Soon after the incubation, the low-pH buffer was replaced with serum-free medium. The cell mixture was then plated onto 384-well plates at a density of 9,000 cells per well, spun down at 220 x g for 2 min, and imaged under a fluorescence microscope using a x10 objective lens. Results were prepared by visually counting three random fields of images from representative experiments and expressed as percentages of the total cells involved in cell-cell fusion. Results are expressed as means standard deviations from a representative exper Innate immune encounter upon virus injection in the capillary vessel. There are several immune-related components circulated in the capillary, which is surrounded by multiple cell layers. The enlarged circle indicates the mosquitos probing site. Prior to the feeding process, mosquito saliva-containing factors (e.g., apyrases) and virus are released into the blood stream Pathogenesis of dengue virus infection. DENV initially infects a cell of the dendritic cell/macrophage/monocyte line (reviewed in reference 5) via receptor-mediated endocytosis and/or enhanced uptake via antibody-virus complexes attached to Fc receptors (reviewed in references 5 and 73). TNF- (22, 52) and NO (24, 141) are produced primarily by infected monocytes/macrophages and activate endothelial cells, which can contribute to increased vascular permeability (reviewed in reference 17). Changes in vascular permeability in DENV infections have classically been measured by monitoring levels of albumin in the plasma (195). IFN- is produced primarily by NK and CD8+ T cells and activates macrophages as well as CD4+ T cells (17). High levels of DENV and sNS1 circulate in the bloodstream (9, 114), and both have been shown to circulate as immune complexes as well. Fc R, Fc gamma receptor Figure 2. Dengue Rash with Sparing of Islands of Skin. Panels A and B show a maculopapular rash with island sparing. Courtesy of Eduardo Gotuzzo and David Freedman, Gorgas Tropical Medicine Course, Universidad Peruana Cayetano Heredia, Lima, Peru.Figure 3. Positive Result on a Tourniquet Test. The tourniquet test is performed by inflating a blood pressure cuff on the upper arm to a point midway between systolic and diastolic blood pressures for five minutes. A test is considered positive when there are 20 or more petechiae per square inch (6.25 cm2) on the forearm. Courtesy of Ann McCarthy, Tropical Medicine and International Health Clinic, Ottawa Hospital, Ottawa.Table 2. Laboratory Diagnosis of Dengue.Figure 2. Kaplan-Meier Curves for Time from Study Entry to Initial (Panel A) and Sustained (Panel B) Cardiovascular Stability among Children in Group 1, According to the Resuscitation Fluid Received. Of 383 patients in group 1,126 received dextran, 129 starch, and 128 Ringer's lactate.Virus-activated signal transduction. Cells respond to virus infection by activating a number of signal-transduction cascades, which lead to nuclear translocation of a specific set of transcription factors. Ultimately, the activated transcription factors stimulate expression of chemokines and other proinflammatory mediators. For a more detailed description of virus-activated signal transduction, see the text. This figure depicts the gene promoter regions of CCL5/RANTES, CCL3/MIP-1 , CXCL8/IL-8, and CXCL10/IP-10, which are discussed in more detail in the text. NF-AT, Nuclear factor of activated T cells; JNK, Jun N-terminal kinase; ATF2, activating transcription factor 2; AP-1, activator protein-1; STAT, signal transducer and activator of transcription; IKK, inhibitor of B (I B) kinase; VAK, virus-activated kinase; IRF, IFN regulatory factor; CRE, cyclic adenosine monophosphate response element; GAS, IFN- activation site; ISRE, IFN-stimulated response element; C/EBP, CCAAT enhancer-binding protein; CD28RE, CD28 response element Model for regulation of VEGF activity by dengue virus and dengue virus-specific immunity. Preexisting nonneutralizing antibody may enhance dengue virus uptake, resulting in a greater viral load. Dengue virus down-regulates the production of sVEGFR2 either directly by interaction with endothelial cells or indirectly via interaction with other cell types. The reduction in sVEGFR2 production results in decreased plasma sVEGFR2 and increased free, biologically active VEGF. VEGF may also be produced by activated dengue virus-specific T cells. A concurrent increase in surface expression of VEGFR2 may enhance endothelial cell responsiveness to VEGF stimulation. The combination of increased biologically active VEGF and enhanced receptor responsiveness results in increased vascular permeability and clinical plasma leakage Vaccines against Dengue and Yellow Fever.Preillness gaps. The innate immune factors between mosquito bites and the appearance of clinical signs are mysterious in dengue disease, either in primary infection or secondary infection. Viremia, IgM, and IgG are all readily detectable during the apparent clinical signs. The oval dotted lines indicate the parameters that are unknown between infection (mosquito bite) and the appearance of illness (day x, normally 2 to 5 days). The solid circles indicate the knowledge gaps in dengue virus infections, primary or secondary. Day y indicates viremia periods, which last from 5 to 7 days. Defervescence is the period that fever and viremia are recessed. A color figure is available in the online version Role of innate immune parameters in DHF/DSS. The proposed alternate hypothesis on DHF/DSS can potentially divide into two stages, disease induction or defined stage and protective stage. Innate immune-related parameters, such as other factors (cytokines, etc.), natural IgM, or platelets, may directly or indirectly contribute to define the development of DHF/DSS during the course of early dengue virus infection (purple circle, preillness). While in the protective stage, the periods in which fever and viral load are recessed, DHF/DSS may attribute to antigen-induced factors, such as IgG and adaptive immune response (blue circle).The purple arrow indicates the time in which, at or around the peak viremia stage, patients reported to the clinics or hospitals, normally after 2 or 3 days of fever. The orange curve and arrow indicate the typical kinetic of viremia. x and y are defined as in Figure 3. Development of an image-based immunofluorescence assay for detection of DENV. (A) Detection of DENV infection of Vero cells using immunofluorescence staining for DENV envelope protein (Env) in the absence and presence of MPA. (B) Dosage-dependent inhibition of DENV 2 infection of cells treated with MPA.Antiviral activity of dasatinib, an inhibitor of c-Src and Abl kinases, on DENV infection. (A) Dosage dependent inhibition of DENV2 infection was observed in C6/36, Vero, and Huh-7 cells. (B) Viral reduction immunofluorescence assays were performed for the indicated viruses to determine the dose-dependent anti-DENV activity of dasatinib.Inhibition of viral spread in dasatinib-treated, DENV-infected cells. An immunofluorescence assay was conducted to detect the localization of the viral envelope (E) protein in DENV-infected Vero, Huh-7, and C6/36 cells that were treated with 2.5 M of dasatinib or DMSO. Noninfected cells are indicated by arrowheads, and accumulation of viral E protein in the perinuclear region is indicated by arrows. Cell nuclei are stained blue with DAPI.siRNA Knockdown of c-Src protein levels inhibits DENV infection. Huh-7 cells were transfected with different concentrations of a c-Src-specific siRNA pool or control siRNA. (A) Knockdown of c-Src protein expression was confirmed by Western blot analysis by using a c-Src specific antibody. Lane 1, Control (siGlo RNA-300 nM); lane 2, c-Src specific siRNA pool200 nM; lane 3, c-Src specific siRNA pool300 nM. Detection of GAPDH was included as a well loading control. (B) DENV2 infection was reduced by >1.5-log units in cells transfected with 200 nM of the c-Src siRNA pool but was unchanged in the presence of 200 nM siGlo or 200 nM GAPDH SMARTpool negative control reagents.Inhibition of c-Src protein kinase activity prevents the assembly of DENV within the ER lumen. DENV2-infected Vero cells treated with either DMSO or 2.5 M of dasatinib (A and B, respectively) and mock-infected cells treated with DMSO (C) were processed for electron microscopy at 4 days postinfection. (A) Representative DENV particles localized within the ER lumen are indicated by arrows. (B) Extensive proliferation of virus-induced membranes is indicated by arrowheads and nucleocapsid-like particles are noted by the arrows. No virus particles are observed within the ER lumen and membranous structures. (C) Typical ultrastructural morphology of noninfected Vero cells. (Scale bars: 200 nm for AC.)