Abstracts

DENGUE

Diagnosis, Diagnostics, Immunodiagnosis & Immunodiagnostics:

January 2003

5986. Chang HH, Shyu HF, Wang YM, Sun DS, Shyu RH, Tang SS, Huang YS. Facilitation of cell adhesion by immobilized dengue viral nonstructural protein 1 (NS1): arginine-glycine-aspartic acid structural mimicry within the dengue viral NS1 antigen. J Infect Dis 2002 Sep 15;186(6):743-51

Dengue virus infection causes life-threatening hemorrhagic fever. Increasing evidence implies that dengue viral nonstructural protein 1 (NS1) exhibits a tendency to elicit potentially hazardous autoantibodies, which show a wide spectrum of specificity against extracellular matrix and platelet antigens. How NS1 elicits autoantibodies remains unclear. To address the hypothesis that NS1 and matrix proteins may have structural and functional similarity, cell-matrix and cell-NS1 interactions were evaluated using a cell-adhesion assay. The present study showed that dengue NS1 immobilized on coverslips resulted in more cell adhesion than did the control proteins. This cell adhesion was inhibited by peptides containing arginine-glycine-aspartic acid (RGD), a motif important for integrin-mediated cell adhesion. In addition, anti-NS1 antibodies blocked RGD-mediated cell adhesion. Although there is no RGD motif in the NS1 protein sequence, these data indicate that RGD structural mimicry exists within the NS1 antigen.

5987. Drosten C, Gottig S, Schilling S, Asper M, Panning M, Schmitz H, Gunther S. Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR. J Clin Microbiol 2002 Jul;40(7):2323-30

Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were horoughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The >or=95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.

5987. Endy TP, Nisalak A, Chunsuttiwat S, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA. Spatial and temporal circulation of dengue virus serotypes: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 2002 Jul 1;156(1):52-9

Dengue virus occurs as four distinct serotypes, each of which causes epidemics throughout the tropical and subtropical regions of the world. Few studies have examined co-circulation of multiple dengue virus serotypes in a well-defined cohort population over time and their capacity to produce severe dengue disease. In this paper, the authors report the details and findings of the first 3 years (1998-2000) of an ongoing prospective study of dengue virus transmission and disease severity in a cohort of children in northern Thailand. A total of 108 dengue virus isolates were obtained from 167 acute dengue virus infections; 23% were DEN-1, 35% were DEN-2, 41% were DEN-3, and 1% were DEN-4. Despite the proximity of the schools, there was marked spatial and temporal clustering of transmission of each dengue serotype. Serotype-specific antibody levels prior to the dengue transmission season were not predictive of the incidence of dengue virus infections or the predominant serotype transmitted at individual schools. All dengue serotypes produced severe dengue illness, although DEN-3 produced more severe symptoms than the other dengue serotypes. The authors' findings emphasize the complexity of dengue serotype-specific virus transmission and severe dengue disease and have important implications for dengue control and vaccine development.

5988. Garcia-Rivera EJ, Rigau-Perez JG. Encephalitis and dengue. Lancet 2002 Jul 20;360(9328):261 No abstract.

5989. Jacobs M, Levin M. An improved endothelial barrier model to investigate dengue haemorrhagic fever. J Virol Methods 2002 Jul;104(2):173-85

A cell culture model suitable for studies of dengue haemorrhagic fever was developed, based on culture of primary human umbilical vein endothelial cells (HUVECs) on a permeable membrane. By electron microscopy, cultured HUVECs at day 11 resembled morphologically microvascular endothelium. Endothelial barrier function was assessed by measuring transendothelial flux of albumin. Instead of using a labelled tracer molecule, an enzyme-linked immunosorbent assay (ELISA) was developed to measure concentrations of native human albumin. The permeability characteristics of the HUVEC monolayer were found to be improved significantly (approximately 1 log reduction in permeability coefficient for albumin) by culturing HUVECs in human serum rather than fetal calf serum. Permeability coefficients for albumin in the range 1-4 x 10(-7) cm/s were achieved, which is an improvement on previous in vitro models of the endothelial barrier. Comparison of transendothelial flux of albumin and urea provided evidence of molecular sieving by the HUVEC monolayer. Moreover, tumour necrosis factor-alpha induced a dose-dependent, reversible increase in permeability of the HUVEC monolayer. This endothelial barrier model thus has many important characteristics that resembled human microvascular endothelium and is an improvement on the previous model proposed for studies of dengue haemorrhagic fever.

5990. King CA, Anderson R, Marshall JS. Dengue virus selectively induces human mast cell chemokine production. J Virol 2002 Aug;76(16):8408-19

Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1alpha, and MIP-1beta, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P < 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1alpha, or MIP-1beta response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.

5991. Rahman M, Rahman K, Siddque AK, Shoma S, Kamal AH, Ali KS, Nisaluk A, Breiman RF. First outbreak of dengue hemorrhagic fever, Bangladesh. Emerg Infect Dis 2002 Jul;8(7):738-40

During the first countrywide outbreak of dengue hemorrhagic fever in Bangladesh, we conducted surveillance for dengue at a hospital in Dhaka. Of 176 patients, primarily adults, found positive for dengue, 60.2% had dengue fever, 39.2% dengue hemorrhagic fever, and 0.6% dengue shock syndrome. The Dengue virus 3 serotype was detected in eight patients.

5992. Rele MC, Rasal A, Deshpande SD, Koppikar GV, Lahiri KR. Indian J med Microbiol. 2001; 19(4): 206-7. No abstract.

5993. Van Gorp EC, Setiati TE, Mairuhu AT, Suharti C, Cate Ht H, Dolmans WM, Van Der Meer JW, Hack CE, Brandjes DP. Impaired fibrinolysis in the pathogenesis of dengue hemorrhagic fever. J Med Virol 2002 Aug;67(4):549-54

The mechanisms contributing to bleeding complications in dengue hemorrhagic fever were studied by investigating the pattern of activation of the coagulation and fibrinolytic systems in 50 children with severe dengue hemorrhagic fever. Thirteen patients (26%) died, and activation of coagulation was most pronounced in the deceased group. Fibrinolysis was also activated, but this activation was relatively weak compared with that of coagulation as a result of persistently high plasminogen activator inhibitor levels. Plasminogen activator inhibitor also prevented a switch from the procoagulant to the profibrinolytic state in lethal dengue hemorrhagic fever, which was further enhanced by an acquired protein C deficiency. The present study is the first to demonstrate such a mechanism in a viral infection. This imbalance between coagulation and fibrinolysis may be used as a prognostic marker, but it may also be a target for future therapeutic intervention. Copyright 2002 Wiley-Liss, Inc.

5994. Wills BA, Oragui EE, Stephens AC, Daramola OA, Dung NM, Loan HT, Chau NV, Chambers M, Stepniewska K, Farrar JJ, Levin M. Coagulation abnormalities in dengue hemorrhagic Fever: serial investigations in 167 Vietnamese children with Dengue shock syndrome. Clin Infect Dis 2002 Aug 1;35(3):277-85

The pathophysiological basis of hemorrhage in dengue infections remains poorly understood, despite the increasing global importance of these infections. A large prospective study of 167 Vietnamese children with dengue shock syndrome documented only minor prolongations of prothrombin and partial thromboplastin times but moderate to severe depression of plasma fibrinogen concentrations. A detailed study of 48 children revealed low plasma concentrations of the anticoagulant proteins C, S, and antithrombin III, which decreased with increasing severity of shock, probably because of capillary leakage. Concurrent increases in the levels of thrombomodulin, tissue factor, and plasminogen activator inhibitor type 1 (PAI-1) indicated increased production of these proteins. Thrombomodulin levels suggestive of endothelial activation correlated with increasing shock severity, whereas PAI-1 levels correlated with bleeding severity. Dengue virus can directly activate plasminogen in vitro. Rather than causing true disseminated intravascular coagulation, dengue infection may activate fibrinolysis primarily, degrading fibrinogen directly and prompting secondary activation of procoagulant homeostatic mechanisms.

Pathogenesis:

5996. Blaney JE Jr, Johnson DH, Manipon GG, Firestone CY, Hanson CT, Murphy BR, Whitehead SS. Genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in SCID mice transplanted with human liver cells. Virology. 2002 Aug 15;300(1):125-39.

Mutations that restrict replication of dengue virus have been sought for the generation of recombinant live-attenuated dengue virus vaccines. Dengue virus type 4 (DEN4) was previously grown in Vero cells in the presence of 5-fluorouracil, and the characterization of 1248 mutagenized, Vero cell passaged clones identified 20 temperature-sensitive (ts) mutant viruses that were attenuated (att) in suckling mouse brain (J. E. Blaney, Jr., D. H. Johnson, C. Y. Firestone, C. T. Hanson, B. R. Murphy, and S. S. Whitehead, 2001, J. Virol. 75(20), 9731-9740). The present investigation has extended these studies by identifying an additional 22 DEN4 mutant viruses which have a small plaque size (sp) phenotype in Vero cells and/or the liver cell line, HuH-7. Five mutant viruses have a sp phenotype in both Vero and HuH-7 cells, three of which are also ts. Seventeen mutant viruses have a sp phenotype in only HuH-7 cells, 13 of which are also ts. Each of the sp viruses was growth restricted in the suckling mouse brain, exhibiting a wide range of reduction in replication (9- to 100,000-fold). Complete nucleotide sequence was determined for the 22 DEN4 sp mutant viruses, and nucleotide substitutions were found in the 3'-untranslated region (UTR) as well as in all coding regions except NS4A. Identical mutations have been identified in multiple virus clones, suggesting that they may be involved in the adaptation of DEN4 virus to efficient growth in Vero cells. Six of the 22 sp 5-FU mutant viruses lacked coding mutations in the structural genes, and 17 recombinant DEN4 viruses were generated which separately encoded each of the mutations observed in these six sp viruses. Analysis of the recombinant DEN4 viruses defined the genetic basis of the sp, ts, and att phenotypes observed in the six sp viruses. Mutations in NS1, NS3, and the 3'-UTR were found to confer a greater than 100-fold, 10,000-fold, and 1000-fold reduction in replication of rDEN4 virus in SCID mice transplanted with HuH-7 cells, respectively, which serves as a novel small animal model for DEN4 infection.

5997. Caplen NJ, Zheng Z, Falgout B, Morgan RA. Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. Mol Ther. 2002 Aug;6(2):243-51.

Mosquitoes transmit numerous viral pathogens to humans including dengue virus which affects approximately 50 million individuals per year. Inhibition of viral gene expression within an insect host could be used to block virus replication and subsequent transmission of the pathogen to humans. A naturally occurring gene silencing mechanism triggered by double-stranded RNA (dsRNA), RNA interference (RNAi), has recently been described in a number of species including Drosophila. To ascertain if dsRNA-triggered RNAi is present in mosquito cells, we used Aedes albopictus C6/36 cells, and to investigate the feasibility of blocking viral gene expression and replication, we used two mosquito-borne viruses, Semliki Forest virus (SFV) and the serotype 1 dengue virus (DEN1). We demonstrate that dsRNA can specifically inhibit transgene expression in C6/36 cells from both plasmid and SFV replicons and can significantly modify the kinetics of DEN1 RNA and virus replication. The inhibition mediated by dsRNA was sequence-specific and either equal or superior to that induced by antisense single-stranded RNA (ssRNA). This study demonstrates dsRNA-triggered inhibition of gene expression and virus replication in mosquito cells and suggests that this mechanism could be used to block pathogen replication within an insect host and, thus, block disease transmission.

5998. Hales S, de Wet N, Maindonald J, Woodward A. Potential effect of population and climate changes on global distribution of dengue fever: an empirical model. Lancet. 2002 Sep 14;360(9336):830-4.

BACKGROUND: Existing theoretical models of the potential effects of climate change on vector-borne diseases do not account for social factors such as population increase, or interactions between climate variables. Our aim was to investigate the potential effects of global climate change on human health, and in particular, on the transmission of vector-borne diseases. METHODS: We modelled the reported global distribution of dengue fever on the basis of vapour pressure, which is a measure of humidity. We assessed changes in the geographical limits of dengue fever transmission, and in the number of people at risk of dengue by incorporating future climate change and human population projections into our model. FINDINGS: We showed that the current geographical limits of dengue fever transmission can be modelled with 89% accuracy on the basis of long-term average vapour pressure. In 1990, almost 30% of the world population, 1.5 billion people, lived in regions where the estimated risk of dengue transmission was greater than 50%. With population and climate change projections for 2085, we estimate that about 5-6 billion people (50-60% of the projected global population) would be at risk of dengue transmission, compared with 3.5 billion people, or 35% of the population, if climate change did not happen. INTERPRETATION: We conclude that climate change is likely to increase the area of land with a climate suitable for dengue fever transmission, and that if no other contributing factors were to change, a large proportion of the human population would then be put at risk.

5999. Kochel TJ, Watts DM, Halstead SB, Hayes CG, Espinoza A, Felices V, Caceda R, Bautista CT, Montoya Y, Douglas S, Russell KL. Effect of dengue-1 antibodies on American dengue-2 viral infection and dengue haemorrhagic fever. Lancet. 2002 Jul 27;360(9329):310-2.

In Iquitos, Peru, no cases of dengue haemorrhagic fever have been recorded in individuals infected with dengue-1 virus followed by American genotype dengue-2 (American dengue-2) virus. We assayed serum samples collected in Iquitos that tested positive for antibodies of monotype dengue-1 and monotype dengue-2 using a plaque reduction neutralisation test to determine their ability to neutralise the infectivity of two dengue-1 viruses, two American dengue-2 viruses, and two Asian dengue-2 viruses. Sera positive for the dengue-1 antibody neutralised dengue-1 viruses and American dengue-2 viruses much more effectively than Asian dengue-2 viruses. Neutralisation of American dengue-2 virus by sera positive for dengue-1 antibodies may account for the absence of dengue haemorrhagic fever in individuals infected with dengue-1 in 1990-91 followed by American dengue-2 virus in 1995 in Iquitos, Peru.

6000. Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM, Chen SH, Liu CC, Chiu SC, Lin YS. Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol. 2002 Jul 15;169(2):657-64.

The onset of vascular leakage and hemorrhagic diathesis is one of the life-threatening complications occurring in dengue patients, yet the pathogenic mechanisms are not well understood. In this study, we demonstrated that Abs against dengue virus nonstructural protein 1 (NS1) generated in mice cross-reacted with human endothelial cells and mouse vessel endothelium. After binding, mouse anti-NS1 Abs induced endothelial cell apoptosis in a caspase-dependent manner. Inducible NO synthase expression could be observed; it showed a time- and dose-dependent correlation with NO production. Endothelial cell apoptosis, characterized by exposure of phosphatidylserine on the cell surface and nuclear DNA fragmentation, was blocked by treatment with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Further studies demonstrated that the expression of Bcl-2 and Bcl-x(L) decreased in both mRNA and protein levels, whereas p53 and Bax increased after anti-NS1 treatment. Cytochrome c release was also observed. All of these effects could be inhibited by N(omega)-nitro-L-arginine methyl ester. Taken together, anti-NS1 Abs act as autoantibodies that cross-react with noninfected endothelial cells and trigger the intracellular signaling leading to the production of NO and to apoptosis. Endothelial cell damage may cause vascular leakage that contributes to the pathogenesis of dengue disease.

6001. Perelygin AA, Scherbik SV, Zhulin IB, Stockman BM, Li Y, Brinton MA. Positional cloning of the murine flavivirus resistance gene. Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9322-7.

Inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genus Flavivirus, which includes human pathogens such as yellow fever, Dengue, and West Nile virus. A single gene, designated Flv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. A positional cloning strategy was used to identify 22 genes from the Flv gene interval including 10 members of the 2'-5'-oligoadenylate synthetase gene family. One 2'-5'-oligoadenylate synthetase gene, Oas1b, was identified as Flv by correlation between genotype and phenotype in nine mouse strains. Susceptible mouse strains produce a protein lacking 30% of the C-terminal sequence as compared with the resistant counterpart because of the presence of a premature stop codon. The Oas1b gene differs from all the other murine Oas genes by a unique four-amino acid deletion in the P-loop located within the conserved RNA binding domain. Expression of the resistant allele of Oas1b in susceptible embryo fibroblasts resulted in partial inhibition of the replication of a flavivirus but not of an alpha togavirus.

Therapy:

6002. Johnson O. Venezuela launches new campaign against dengue fever. BMJ 2002 Sep 7;325(7363):512 No abstract.

6003. Teixeira Mda G, Barreto ML, Costa Mda C, Ferreira LD, Vasconcelos PF, Cairncross S. Dynamics of dengue virus circulation: a silent epidemic in a complex urban area. Trop Med Int Health 2002 Sep;7(9):757-62

Serotypes of dengue DEN-1 and DEN-2 have been reported in much of Brazil over the last 15 years, and DEN-3 serotype was only recently detected. This - prospective study was conducted in Salvador, a large city in north-east Brazil, where two epidemics were previously recorded (DEN-1 and DEN-2). We obtained the seroprevalence and 1-year incidence of dengue infections in the population of 30 sampling areas of Salvador and analysed the relationship between intensity of viral circulation, standard of living and vector density. High seroprevalence (68.7%) and annual incidence (70.6%) of infection for one or both circulating serotypes (DEN-1 and DEN-2) were found. High rates of transmission were observed in all studied areas, from the highest to the lowest socio-economic status. The mean PI (Premise Index) for Aedes aegypti was 7.4% (range 0.27-25.6%). Even in the areas with the lowest PI (</=3%) the observed seroincidence was 54.6%. These findings highlighted the existence of a silent epidemic during a period perceived by the Health Services as of low endemicity, indicating the strength and speed of dengue transmission in the city of Salvador.

April 2003

6634. Arora MM; Mishra KB; Nair V; Bhardwaj JR; Bhalwar R; Somani BL. Diagnosing disseminated intravascular coagulation in acute infection : can we do without FDP & D-DIMER. Medical Journal Armed Forces India. 2002 Jan; 58(1): 13-7.

ABSTRACT: Alterations in coagulation profile viz. platelet count, prothrombin time (PT), partial thromboplastin time with kaolin (PTTK), thrombin time (TT) and fibrinogen were studied in 96 patients (73 males and 23 females) of acute infections. Fibrin/fibrinogen degradation products (FDP) level 25miug fibrinogen equivalent unit (FEU)/ml along-with D-dimer 1.0miug FEU/ml was considered criteria for diagnosis of disseminated intravascular coagulation (DIC). Normal values were established using plasma from 12 healthy voluntary blood donors. Out of these 96 patients, 15 had infection with Gram positive bacteria, 23 with Gram negative bacteria and 38 with Dengue. In 20 patients, nature of infection was not defined. Mean platelet count per cubic millimeter was 2.14 lac in Gram positive infection and 1.74 lac in Gram negative infection (p=0.07). There was no significant difference in other coagulation parameters in Gram positive and Gram negative infection. Platelet counts were low in 71 percent of Dengue patients but there was no significant alteration in PT, PTTK and TT. None of the Dengue patients had hypofibrinogenemia or DIC though hyperfibrinogenemia was present in 21 percent of Dengue patients. 20 patients had features of septicemia (Gram +ve 7, Gram-ve 8, undefined 5); 10 had concomitant DIC. DIC was present in additional 4 patients of acute infection without septicemia. PTTK was raised in 60 percent of the septicemia patients. 20 out of 82 non- DIC acute infection patients had subnormal PTTK. Commonest alteration in 14 DIC patients was raised PTTK with a sensitivity of 78.6 percent and specificity of 81.7 percent. Low fibrinogen levels though specific for DIC, were present in only 21.4 percent of the DIC patients. Combinations of PTTK 38 sec with PT15 sec or platelet count 1.5 lac/mm cube were good screening tests for DIC and detected 11 and 10 patients out of 14 with three and two false positives respectively.

6635. Dutta P , Khan S A , Khan A M , Sharma C K , Doloi P K , Mahant J . Soloid waste pollution and breeding Potential of dengue vector in urban and industrial environment of Assam J envir biol ,Lucknow 1999,20(4),343-5. (ISA 014219, Vol 38 No14 ,16 July 2002)

6636. Libraty DH, Young PR, Pickering D, Endy TP, Kalayanarooj S, Green S, Vaughn DW, Nisalak A, Ennis FA, Rothman AL. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002 Oct 15;186(8):1165-8

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (> or =600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.

Pathogenesis:

6637. Lin YL, Lei HY, Lin YS, Yeh TM, Chen SH, Liu HS. Heparin inhibits dengue-2 virus infection of five human liver cell lines. Antiviral Res 2002 Oct;56(1):93-6

Liver is suggested to be the major target of dengue virus infection and plays an important role in the immunopathogenesis of dengue hemorrhagic fever. Previously, we reported that five human liver cell lines (HuH-7, HA22T, Hep3B, PLC, and Chang liver) with various degrees of differentiation and tumorigenicity showed different susceptibility for dengue virus infection. Here, we demonstrate that heparin, an analogue of heparan sulfate (HS), can compete with HS on cell membrane for virus binding and subsequently inhibits the replication of dengue-2 and Japanese encephalitis viruses in hepatoma and BHK-21 cells, respectively. It indicates that the binding of these viruses with HS is an important process for their invasion. Moreover, the inhibitory effect of heparin correlates with the infectivity of the virus in the cells. All together, our results suggest that HS is an important host component for dengue and Japanese encephalitis virus replication, which can be effectively blocked by heparin.

6638. Lin YW, Wang KJ, Lei HY, Lin YS, Yeh TM, Liu HS, Liu CC, Chen SH. Virus replication and cytokine production in dengue virus-infected human B lymphocytes. J Virol 2002 Dec;76(23):12242-9

Dengue virus (DV) replication, antibody-enhanced viral infection, and cytokine responses of human primary B lymphocytes (cells) were characterized and compared with those of monocytes. The presence of a replication template (negative-strand RNA intermediate), viral antigens including core and nonstructural proteins, and increasing amounts of virus with time postinfection indicated that DV actively replicated in B cells. Virus infection also induced B cells to produce interleukin-6 and tumor necrosis factor alpha, which have been previously implicated in virus pathogenesis. In addition, a heterologous antibody was able to enhance both virus and cytokine production in B cells. Furthermore, the levels of virus replication, antibody-enhanced virus replication, and cytokine responses observed in B cells were not statistically different from those in monocytes. These results suggest that B cells may play an important role in DV pathogenesis.

Vaccines:

6639. Halstead SB, Deen J. The future of dengue vaccines. Lancet 2002 Oct 19;360(9341):1243-5. No abstract.

Therapy:

6640. Van Benthem BH, Khantikul N, Panart K, Kessels PJ, Somboon P, Oskam L. Knowledge and use of prevention measures related to denguen northern Thailand. Trop Med Int Health 2002 Nov;7(11):993-1000

OBJECTIVE: To determine the frequency and determinants of knowledge of dengue infection in three sites in northern Thailand, and to compare prevention measures of people with and without knowledge of dengue. METHODS: In May 2001 we conducted an epidemiological survey among 1650 persons living in three areas in northern Thailand. Knowledge of dengue and the use of prevention measures were measured by means of a structured questionnaire. Differences in knowledge of dengue and the use of prevention measures between risk groups were calculated by chi-square test. Logistic regression was used to identify determinants of knowledge. RESULTS: Of the 1650 persons, 67% had knowledge of dengue. Fever (81%) and rash (77%) were the most frequently mentioned symptoms. Persons with knowledge of dengue reported a significantly higher use of prevention measures than persons without knowledge of dengue. In multivariate analyses, knowledge of dengue significantly differed by age, sex, occupation and site (P < 0.05). Younger people knew more about dengue than older persons: adjusted odds ratio (aOR) of 6.75 [95% confidence interval (CI): 4.32-10.6] for the 15-29 age group compared with people aged 60 and older. In comparison with farmers (reference group), knowledge of dengue was significantly higher among students (aOR: 10.6, 95% CI: 4.27-26.4), but lower among housewives or unemployed persons (aOR: 0.44, 95% CI: 0.31-0.64). CONCLUSION: The overall knowledge of dengue was high, but housewives, unemployed and old persons had relatively little knowledge of dengue. Therefore, these groups may need special attention in future dengue education programmes. Persons with knowledge of the disease more frequently reported the use of preventive measures, indicating the value of education programmes as a tool in dengue prevention.

July 2003

7057. Espina LM, Valero NJ, Hernandez JM, Mosquera JA. Increased apoptosis and expression of tumor necrosis factor-alpha caused by infection of cultured human monocytes with dengue virus. Am J Trop Med Hyg 2003 Jan;68(1):48-53

Dengue (DEN) virus is responsible for one of the most significant viral diseases in tropical countries. Monocytes/macrophages (Mo/Mphi) are the major target cells for DEN virus. To determine the effects of the interaction between DEN virus and Mo/Mphi, human monocyte cultures were infected with DEN virus type 2. Apoptosis and production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured in control and infected cultures. Virus was taken up by phagocytosis, but no membrane-coated pits at the virus attachment sites were observed. Increased number of apoptotic cells and increased production of TNF-a were observed in infected monocyte cultures. No increase in production of nitric oxide was observed. These results may be related to early primary viral infection, in which virus could induce apoptosis in monocytes, but monocytes may contribute to host defense mechanisms against virus by viral phagocytosis, phagocytosis of infected apoptotic cells, and the release of proinflammatory cytokines.

7058. Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS, Yeh TM, Chen SH, Lin YS. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol 2003 Jan;69(1):82-90

Dengue virus infection causes a wide range of diseases from the mild febrile illness dengue fever to the life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage and hemorrhagic syndrome are the clinical features associated with dengue infection, yet the mechanisms remain unclear. In this study, the cross-reactivity of dengue patient sera with endothelial cells was demonstrated. There were higher percentages of endothelial cells reactive with dengue hemorrhagic fever/dengue shock syndrome patient sera than those with dengue fever patient sera. The percentages of endothelial cells reactive with patient serum IgM were higher than those with IgG. Further studies showed that the endothelial cell binding activity was inhibited by pretreatment with dengue virus nonstructural protein 1 (NS1). The antibodies against NS1 produced after dengue virus infection may, at least in part, account for the cross-reactivity of patient sera with endothelial cells. Furthermore, dengue patient sera induced endothelial cell apoptosis via a caspase-dependent pathway that was also inhibited by NS1 pretreatment. In addition to apoptosis, patient sera caused cell lysis in the presence of complement, and DHF/DSS patient sera showed higher percentages of cytotoxicity than dengue fever patient sera. Thus, the generation of cross-reactive autoantibodies against endothelial cells would lead to their dysfunction, which may play a role in the pathogenesis of dengue virus infection. Copyright 2003 Wiley-Liss, Inc.

7059. Mairuhu AT, Mac Gillavry MR, Setiati TE, Soemantri A, ten Cate H, Brandjes DP, van Gorp EC. Is clinical outcome of dengue-virus infections influenced by coagulation and fibrinolysis? A critical review of the evidence. Lancet Infect Dis 2003 Jan;3(1):33-41

Despite efforts to elucidate the pathogenesis of dengue fever, the progression into severe disease remains poorly understood. In-vitro findings suggest that coagulopathy and disturbances in fibrinolysis have a pivotal role in the pathophysiology. If disturbances in these processes are predictive of clinical outcome in this disease, there could be important consequences for both diagnosis and treatment. We have critically reviewed publications on this topic to assess whether there is an association between activation of coagulation and fibrinolysis and clinical outcome of dengue-virus infections. In general, the selected studies showed activation of both the coagulation and fibrinolytic systems in this infection. The activation was more pronounced in severe infections and in cases with a poor clinical outcome. However, the findings were not consistent, and owing to a lack of detailed information on characteristics of patients, disease, and study design, we could not ascertain whether inconsistencies were caused by differences in these characteristics, selection bias, or confounding factors. We conclude that an association between activation of coagulation and fibrinolysis and clinical outcome of dengue-virus infections is conceivable but has been inadequately assessed and that methodologically sound studies, complemented with complete and reliable reporting, are needed to show whether there is a true association.

7060. Yocupicio-Monroy RM, Medina F, Reyes-del Valle J, del Angel RM. Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA. J Virol 2003 Mar;77(5):3067-76

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.

Pathogenesis:

7061. Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS, Yeh TM, Chen SH, Lin YS. Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol. 2003 Jan;69(1):82-90.

7062. Oliveira SA, Siqueira MM, Camacho LA, Castro-Silva R, Bruno BF, Cohen BJ. Use of RT-PCR on oral fluid samples to assist the identification of measles cases during an outbreak. Epidemiol Infect. 2003 Feb;130(1):101-6.

This study investigated the occurrence of mild modified measles cases during an outbreak in Niteroi, RJ, Brazil by using RT-PCR on oral fluid samples. From August to December 1997 a total of 76 patients with rash were seen at the study sites. Confirmed diagnosis by serology was achieved in 47 cases: measles (39.5%), rubella (13.2%), HHV-6 (3.9%), human parvovirus B19 (3.9%), dengue fever (3%). For 19 of the 29 patients without a conclusive diagnosis paired serum and saliva samples were available for further tests. In four of them, measles virus RNA was dete cted by RT-PCR in saliva samples in the absence of specific IgM in serum samples. Vaccination histories obtained from three of the RT-PCR positive cases showed that individuals previously immunized can still be infected and contribute to the circulation of measles virus. This study demonstrated the usefulness of RT-PCR on non-invasive clinical samples for the investigation of measles cases.

7063. Suksanpaisan L, Smith DR. Analysis of saturation binding and saturation infection for dengue serotypes 1 and 2 in liver cells. Intervirology. 2003;46(1):50-5.

OBJECTIVE: The liver has been increasingly recognized as a significant target organ in the pathogenesis of dengue virus infection. However, only two contradictory studies have examined the binding of the dengue virus to liver cells. This study therefore sought to investigate the binding of the dengue virus to HepG2 cells. METHODS: Radiolabeled dengue virus serotypes 1 and 2 were prepared through viral propagation in Vero cells. Increasing amounts of virus were then incubated with HepG2 cells to determine the ability of the virus to achieve saturation of binding on HepG2 cells. RESULTS: Results indicated that it was not possible to reach saturation of binding under experimentally achievable conditions. We then sought to determine whether it was possible to reach a state of saturation of infection, by using increasingly high titers of virus on a constant number of cells. Dengue serotype 1 showed no evidence of saturation of infection, even at titers of 5,000 viruses per cell. In contrast, dengue serotype 2 became saturated at levels of approximately 3,000 viruses per cell. CONCLUSIONS: These results are consistent with proposals that dengue virus binding to cells is mediated initially through a low-affinity interaction with an abundant molecule on the surface of the cell and secondly through interaction with a less commonly expressed molecule, which is required for viral internalization. Copyright 2003 S. Karger AG, Basel

7064. Yocupicio-Monroy RM, Medina F, Reyes-del Valle J, del Angel RM. Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA. J Virol. 2003 Mar;77(5):3067-76.

October 2003

7859. Imbert-Laurenceau E, Crepinior J, Crance JM, Jouan A, Migonney V. Polystyrene derivatives substituted with arginine interact with Babanki (Togaviridae) and Kedougou (Flaviviridae) viruses. J Med Virol. 2003 Apr;69(4):503-9.

Outbreaks of new or old diseases appear primarily in tropical zones such as Africa, south and central America, or Asia. Among these diseases, those induced by Arboviruses (the best known of which are being yellow fever, dengue, Ebola, and Sindbis) are under intensive observation by the World Health Organization. Rapid isolation and identification of the viral species is the first step in the diagnosis, study, and control of epidemics. One major problem with the isolation of viruses is capturing sufficient numbers of viral particles to test. The work presented in this report addresses this question. We have tested the interaction between Babanki (Togaviridae), Kedougou (Flaviviridae) viruses, and a range of insoluble polystyrene derivatives substituted with arginine groups. Insoluble functionalized copolymers were found to develop specific interactions with viruses through chemical groups present on their surfaces. The adsorption of viruses varied according to the percentage of arginine substituted onto the polymer, with a maximum value for both viruses of about 20% of grafting rate. It was also found that the Kedougou virus displayed the highest affinity for this polymer. Copyright 2003 Wiley-Liss, Inc.

7860. Koraka P, Murgue B, Deparis X, Setiati TE, Suharti C, van Gorp EC, Hack CE, Osterhaus AD, Groen J. Elevated levels of total and dengue virus-specific immunoglobulin E in patients with varying disease severity. J Med Virol. 2003 May;70(1):91-8.

The kinetics of total and dengue virus-specific immunoglobulin E (IgE) were studied in serial serum samples obtained from 168 patients, 41 of whom suffered from primary dengue virus infection and 127 suffered from secondary dengue virus infection. Seventy-one patients were classified as dengue fever, 30 as dengue hemorrhagic fever, and 67 as dengue shock syndrome. A control group included single serum samples from patients with a herpes virus infection (n = 14), non-dengue febrile patients (n = 10), and healthy blood donors (n = 10). Patients with dengue virus infection had higher levels of total and dengue virus-specific IgE than non-dengue patients (P < 0.05). Patients with secondary dengue virus infections had not significantly increased levels of both total and dengue virus-specific IgE in the acute phase of disease compared to patients with primary dengue virus infections. Dengue virus-specific IgE was significantly higher in dengue hemorrhagic fever and/or dengue shock syndrome patients compared to dengue fever and non-dengue patients (P < 0.05). In conclusion, this study showed elevated total and dengue virus-specific IgE serum antibody levels in the acute stage of disease. Therefore, measurement of both total and dengue virus-specific IgE serum antibodies can be used as an additional prognostic marker in the development of severe complications in dengue virus infections. In addition, the presence and increase of dengue virus-specific IgE serum antibodies in patients with dengue virus infections is suggestive of the pathogenetic role that IgE may play in the hemostatic disorders observed in dengue hemorrhagic fever and dengue shock syndrome.

7861.Shu PY, Chang SF, Kuo YC, Yueh YY, Chien LJ, Sue CL, Lin TH, Huang JH. Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus. J Clin Microbiol. 2003 Jun;41(6):2408-16.

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10(7) PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

7862. Vazquez S, Valdes O, Pupo M, Delgado I, Alvarez M, Pelegrino JL, Guzman MG. MAC-ELISA and ELISA inhibition methods for detection of antibodies after yellow fever vaccination. J Virol Methods. 2003 Jun 30;110(2):179-84.

The IgM antibody capture ELISA (MAC-ELISA) and ELISA inhibition methods for the detection of antibodies against dengue virus were modified to detect antibodies against yellow fever virus. Tests were carried out in 21 persons vaccinated with 17D and compared with the Plaque reduction neutralizing test. Of 17 naïve subjects vaccinated, 16 (94%) seroconverted using the MAC-ELISA test and 14 (82%) seroconverted (or >/=fourfold titer increase) in the ELISA inhibition method. Cross-reactivity was evaluated by both tests and resulted in a high specificity to IgM antibodies against yellow fever, when all the samples from vaccinated individuals were negative by MAC-ELISA using dengue antigen. However, 10.7% of the positive dengue sera from the Santiago de Cuba epidemic cross-reacted by MAC-ELISA using yellow fever antigen. ELISA inhibition method showed high cross-reactivity when the 21 sera pairs were worked with yellow fever and dengue antigens. The MAC-ELISA and ELISA inhibition methods have become indispensable tools in our laboratory in order to maintain a surveillance system for dengue and dengue hemorrhagic fever. They are relatively rapid, simple, and they do not require sophisticated equipment. Both MAC-ELISA and

ELISA inhibition methods for yellow fever could be useful for diagnosis, surveillance and yellow fever vaccine evaluation.

Pathogenesis

7863. Blaney JE Jr, Manipon GG, Murphy BR, Whitehead SS. Temperature sensitive mutations in the genes encoding the NS1, NS2A, NS3, and NS5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice. Arch Virol. 2003 May;148(5):999-1006.

7864. Cologna R, Rico-Hesse R. American genotype structures decrease dengue virus output from human monocytes and dendritic cells. J Virol. 2003 Apr;77(7):3929-38.

7865. Jones CT, Ma L, Burgner JW, Groesch TD, Post CB, Kuhn RJ. Flavivirus capsid is a dimeric alpha-helical protein. J Virol. 2003 Jun;77(12):7143-9.

7866. Koraka P, Murgue B, Deparis X, Setiati TE, Suharti C, van Gorp EC, Hack CE, Osterhaus AD, Groen J. Elevated levels of total and dengue virus-specific immunoglobulin E in patients with varying disease severity. J Med Virol. 2003 May;70(1):91-8.

7867. Tassaneetrithep B, Burgess TH, Granelli-Piperno A, Trumpfheller C, Finke J, Sun W, Eller MA, Pattanapanyasat K, Sarasombath S, Birx DL, Steinman RM, Schlesinger S, Marovich MA. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J Exp Med. 2003 Apr 7;197(7):823-9.

7868. Wei HY, Jiang LF, Xue YH, Fang DY, Guo HY. Secreted expression of dengue virus type 2 full-length envelope glycoprotein in Pichia pastoris. J Virol Methods. 2003 Apr;109(1):17-23.

Vaccines:

7869. Konishi E, Terazawa A, Imoto J. Simultaneous immunization with DNA and protein vaccines against Japanese encephalitis or dengue synergistically increases their own abilities to induce neutralizing antibody in mice. Vaccine. 2003 May 16;21(17-18):1826-32.

Gene-based and protein-based vaccines are two distinct types of vaccines. In this report, we examined if combined use of DNA and protein vaccines would increase their own abilities to induce neutralizing antibody in murine models for Japanese encephalitis (JE) or dengue type 2 (DEN2). DNA vaccines for JE (pcJEME) or DEN2 (pcD2ME) were inoculated intramuscularly, and protein vaccines consisting of subviral extracellular particles (EPs) containing JE (JEEP) or DEN2 (D2EP) virus antigens were inoculated subcutaneously with Freund's adjuvant. Two immunizations of ICR mice with pcJEME and/or JEEP in the prime-boost protocol indicated that levels of neutralizing antibody induced by the pcJEME prime-JEEP boost vaccination were two to eight-fold higher than those induced by pcJEME alone, but were equivalent to those induced by JEEP alone and slightly higher than those induced by the JEEP prime-pcJEME boost regimen. On the other hand, simultaneous immunization of ICR mice with pcJEME and JEEP provided synergistically higher neutralizing antibody titers than those provided by immunization with either immunogen. Immunization with graded doses of pcJEME and JEEP confirmed the synergism. The synergistic increase in neutralizing antibody titer by simultaneous immunization with DNA and protein vaccines was also shown by immunization with pcD2ME and D2EP in ICR and ddY mice. Both IgG1 and IgG2a antibodies were induced by combined immunization with pcJEME and JEEP.

7870. Puttikhunt C, Kasinrerk W, Srisa-ad S, Duangchinda T, Silakate W, Moonsom S, Sittisombut N, Malasit P. Production of anti-dengue NS1 monoclonal antibodies by DNA immunization. J Virol Methods. 2003 Apr;109(1):55-61.

Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 microg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.

Therapy:

7871. Ostronoff M, Ostronoff F, Florencio R, Florencio M, Domingues MC, Calixto R, Sucupira A, Souto Maior AP, Matias C, Matias K, Tagliari C, Soussain C. Serious thrombocytopenia due to dengue hemorrhagic fever treated with high dosages of immunoglobulin. Clin Infect Dis. 2003 Jun 15;36(12):1623-4. No abstract

7872. Puttikhunt C, Kasinrerk W, Srisa-ad S, Duangchinda T, Silakate W, Moonsom S, Sittisombut N, Malasit P. Production of anti-dengue NS1 monoclonal antibodies by DNA immunization. J Virol Methods. 2003 Apr;109(1):55-61.

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