Immunoglobulin M enzyme-linked immunosorbent assay using recombinant polypeptides for diagnosis of dengue

We demonstrate that a mixture of four recombinant dengue virus E polypeptides corresponding to the N-terminal region of the envelope protein from all serotypes substitutes for standard antigens in two immunoglobulin M enzyme-linked immunosorbent assay formats with 100% concordance, making these polypeptides a useful and accessible reagent for serological diagnosis of dengue in endemic countries.     

Recombinant polypeptide antigen-based immunoglobulin G enzyme-linked immunosorbent assay for serodiagnosis of dengue

We have developed an indirect enzyme-linked immunosorbent assay for detection of anti-dengue virus (DENV) immunoglobulin G antibodies using four recombinant DENV envelope polypeptides as antigens, which demonstrated a sensitivity of 89.4% and a specificity of 93.3%. These easily produced antigens are a feasible, cost-effective alternative for generating reagents for dengue serological tests.     

Computer analysis of antigenic domains and RGD-like sequences (RGWG) in the E glycoprotein of flaviviruses: An approach to vaccine development

Antigenic domains and RGD-like sequences in the E glycoprotein of the flaviviruses Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue type 4 virus, and tick-borne encephalitis virus were analyzed by computer programs that provide information on the physical properties of the polypeptides. The use of computer programs for the development of vaccines based on the synthesis of antigenic peptides is discussed. Synthetic viral peptides are proposed to be used for topical application so as to interfere with the virus-cell interaction. Viral peptides with antigenic epitopes to protect against dengue virus infection without enhancing pathogenesis may also be developed on the basis of the computer analysis.     

Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.     

Evaluation of a commercial dengue NS1 antigen-capture ELISA for laboratory diagnosis of acute dengue virus infection

A commercial dengue NS1 antigen-capture ELISA was evaluated to demonstrate its potential application for early laboratory diagnosis of acute dengue virus infection. Dengue virus NS1 antigen was detected in 199 of 213 acute serum samples from patients with laboratory confirmation of acute dengue virus infection but none of the 354 healthy blood donors’ serum specimens. The dengue NS1 antigen-capture ELISA gave an overall sensitivity of 93.4% (199/213) and a specificity of 100% (354/354). The sensitivity was significantly higher in acute primary dengue (97.3%) than in acute secondary dengue (70.0%). The positive predictive value of the dengue NS1 antigen-capture ELISA was 100% and negative predictive value was 97.3%.

Comparatively, virus isolation gave an overall positive isolation rate of 68.1% with a positive isolation rate of 73.9 and 31.0% for acute primary dengue and acute secondary dengue, respectively. Molecular detection of dengue RNA by RT-PCR gave an overall positive detection rate of 66.7% with a detection rate of 65.2 and 75.9% for acute primary dengue and acute secondary dengue, respectively.

The results indicate that the commercial dengue NS1 antigen-capture ELISA may be superior to virus isolation and RT-PCR for the laboratory diagnosis of acute dengue infection based on a single serum sample.     

Evaluation of a Rapid Immunochromatographic Test for Diagnosis of Dengue Virus Infection

A rapid (<7-min) immunochromatographic test for immunoglobulin M (IgM) and IgG antibodies to dengue viruses was evaluated by using hospital admission and discharge sera from 124 patients. The reference laboratory diagnosis was based on the results of virus isolation, hemagglutination-inhibition assay (HAI), and enzyme immunoassay (EIA). By the standard assays, patients experienced primary dengue virus infection (n = 30), secondary dengue virus infection (n = 48), Japanese encephalitis (JE) virus infection (n = 20), or no flavivirus infection (n = 26). The rapid test demonstrated 100% sensitivity in the diagnosis of dengue virus infection and was able to distinguish between primary and secondary dengue virus infections through the separate determinations of IgM and IgG. For all patients with primary dengue virus infection a positive test for IgM to dengue virus and a negative test for IgG to dengue virus were obtained, whereas for 46 of 48 patients (96%) with secondary dengue virus infection, a positive test for IgG to dengue virus with or without a positive test for IgM to dengue virus was obtained. The remaining two patients with secondary dengue virus infection had positive IgM test results and negative IgG test results. Furthermore, the rapid test was positive for patients confirmed to be infected with different dengue virus serotypes (12 infected with dengue virus serotype 1, 4 infected with dengue virus serotype 2, 3 infected with dengue virus serotype 3, and 2 infected with dengue virus serotype 4). The specificity of the test for nonflavivirus infections was 88% (3 of 26 positive), while for JE virus infections the specificity of the test was only 50% (10 of 20). However, most patients with secondary dengue virus infection were positive for both IgM and IgG antibodies to dengue virus, while no patients with JE virus infection had this profile, so cross-reactivity was only a concern for a small proportion of patients with secondary dengue infections. The rapid test demonstrated a good correlation with the reference EIA and HAI and should be useful for the rapid diagnosis of dengue virus infections.     

Single rapid TaqMan fluorogenic probe based PCR assay that detects all four dengue serotypes

Public health laboratories require rapid diagnosis of dengue outbreaks for application of measures such as vector control. We have developed a rapid single fluorogenic probe-based polymerase chain reaction assay for the detection of all four dengue serotypes (FUDRT-PCR). The method employs primers and probe that are complementary to the evolutionarily conserved 3' untranslated region of the dengue genome. The assay detected viral RNA of strains of all four dengue serotypes but not of the flaviviruses Japanese encephalitis virus, Murray Valley encephalitis virus, Kunjin, Stratford, West Nile, Alfuy or Yellow fever. When compared to an existing nested-PCR assay for the detection of dengue on clinical samples, FUDRT-PCR detected dengue 1 (100%, n=14), dengue 2 (85%, n=13), dengue 3 (64%, n=14) and dengue 4 (100%, n=3) with the indicated sensitivities. FUDRT-PCR enables diagnosis of acute dengue infection in four hours from sample receipt. In addition, a single-test procedure should result in a reduction in the number of tests performed with considerable cost savings for diagnostic laboratories.     

Rapid subtyping of dengue virus serotypes 1 and 4 by restriction site-specific PCR

We previously reported a simple subtyping method, restriction site-specific PCR (RSS-PCR), for dengue virus serotypes 2 and 3; here we describe its application for subtyping dengue virus serotypes 1 and 4. Three major RSS-PCR types were observed for dengue virus serotype 1 and two types were observed for dengue virus serotype 4, in agreement with previous strain classifications based on sequence analysis. Because of its simplicity, this method is amenable to rapid subtyping and application to epidemiological studies of dengue in countries where dengue is endemic.     

Role of T cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever

Dengue infections are a major cause of morbidity and mortality in the tropical and sub-tropical regions of the world. There is no vaccine for dengue and also there are no anti-viral drugs to treat the infection. Some patients, typically those experiencing a secondary infection with a different dengue serotype, may progress from an acute febrile disease to the more severe forms of disease, dengue haemorrhagic fever and dengue shock syndrome. Here we discuss the significant immunopathological component to severe disease and how T cells, cytokines and cross-reactive antibody combine to contribute to the progression to dengue haemorrhagic fever. These events are thought to lead to vascular leakage, the signature event in dengue haemorrhagic fever, and are addressed in this review by incorporating the concept of heterologous T cell immunity. The need for effective measures against dengue and dengue-related illness is clear. We propose that drugs against dengue virus, or the symptoms of severe dengue disease, are a viable goal.     

Antibodies from dengue patient sera cross-react with endothelial cells and induce damage

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.     

Serological markers during dengue 3 primary and secondary infections.

BACKGROUND: The detection of the IgM antibody for the dengue virus in serum by ELISA has become one of the most important and useful methods for diagnosis of dengue using a single acute-phase serum sample. Currently, this system is an invaluable tool for the surveillance of dengue fever (DF) and dengue hemorrhagic fever (DHF). The usefulness of other serological markers such as IgA and IgE have been less studied. OBJECTIVE: To study the IgM, IgA and IgE specific antibody response in dengue 3 infected patients with different clinical picture and type of infection. STUDY DESIGN: One hundred and twenty-seven serum samples collected on days 5-7 at the onset of fever from clinically and serologically confirmed dengue cases were studied. Forty-two were classified as primary dengue fever cases, 48 as secondary dengue fever cases and 37 as secondary dengue hemorrhagic fever cases. All samples were tested by capture ELISA in order to detect dengue IgM, IgA and IgE antibodies. RESULTS AND CONCLUSIONS: In this study, significant differences were observed in the IgM, IgA and IgE response between the study groups. High IgA and IgE OD ratios in secondary dengue cases were found. The usefulness of serotype specific IgM antibody detection is also analyzed and discussed. A priority for future dengue research in terms of protection, recovery of infection and immunopathogenesis is to elucidate the role of these immunoglobulins. The cross reactivity response to IgM between dengue virus serotypes in primary and secondary cases should also be more studied.     

Fuzzy model identification of dengue epidemic in Colombia based on multiresolution analysis

ObjectiveThis article presents a model of a dengue and severe dengue epidemic in Colombia based on the cases reported between 1995 and 2011.MethodologyWe present a methodological approach that combines multiresolution analysis and fuzzy systems to represent cases of dengue and severe dengue in Colombia. The performance of this proposal was compared with that obtained by applying traditional fuzzy modeling techniques on the same data set. This comparison was obtained by two performance measures that evaluate the similarity between the original data and the approximate signal: the mean square error and the variance accounted for. Finally, the predictive ability of the proposed technique was evaluated to forecast the number of dengue and severe dengue cases in a horizon of three years (2012–2015). These estimates were validated with a data set that was not included into the training stage of the model.ResultsThe proposed technique allowed the creation of a model that adequately represented the dynamic of a dengue and severe dengue epidemic in Colombia. This technique achieves a significantly superior performance to that obtained with traditional fuzzy modeling techniques: the similarity between the original data and the approximate signal increases from 21.13% to 90.06% and from 18.90% to 76.83% in the case of dengue and severe dengue, respectively. Finally, the developed models generate plausible predictions that resemble validation data. The difference between the cumulative cases reported from January 2012 until July 2013 and those predicted by the model for the same period was 24.99% for dengue and only 4.22% for severe dengue.ConclusionsThe fuzzy model identification technique based on multiresolution analysis produced a proper representation of dengue and severe dengue cases for Colombia despite the complexity and uncertainty that characterize this biological system. Additionally, the obtained models generate plausible predictions that can be used by surveillance authorities to support decision-making oriented to designing and developing control strategies.     

用逆转录-聚合酶链反应检测海南岛登革热流行区某些动物体内登革病毒RNA

目的寻找中国海南岛一带登革热疫区，登革病毒潜伏的动物宿主及鉴定其毒株型别。方法采用１－４型登革病毒通用引物，用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测海南岛登革热流行区蝙蝠脑细胞，血清和埃及伊蚊的登革病毒ＲＮＡ。结果检测３５例疫区蝙蝠脑细胞，２０例阳性；检测１８例蝙蝠血清，３例阳性；检测三组埃及伊蚊，１组阳性，３组非流行区者都阴性。用４个登革病毒原型株的单克隆荧光抗体技术检测２０例登革病毒ＲＮＡ阳性的蝙蝠脑细胞压印片，１６例为２型株阳性，与ＲＴ－ＰＣＲ检测登革热流行区登革病毒ＲＮＡ阳性蝙蝠脑细胞的阳性符合率为８０００％。用ＲＴ－ＰＣＲ检测非流行区蝙蝠脑细胞登革病毒ＲＮＡ，均为阴性。结论本研究证实蝙蝠是登革病毒的贮存宿主，为登革热流行的有效控制提供了一定重要的线索。     

Dengue vaccines: Challenges,development, current status and prospects

Infection with dengue virus (DENV) is the most rapidly spreading mosquito-borne viral disease in the world. The clinical spectrum of dengue, caused by any of the four serotypes of DENV, ranges from mild self-limiting dengue fever to severe dengue, in the form dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Increased rates of hospitalization due to severe dengue, during outbreaks, result in massive economic losses and strained health services. In the absence of specific antiviral therapy, control of transmission of DENV by vector management is the sole method available for decreasing dengue-associated morbidity. Since vector control strategies alone have not been able to satisfactorily achieve reduction in viral transmission, the implementation of a safe, efficacious and cost-effective dengue vaccine as a supplementary measure is a high public health priority. However, the unique and complex immunopathology of dengue has complicated vaccine development. Dengue vaccines have also been challenged by critical issues like lack of animal models for the disease and absence of suitable markers of protective immunity. Although no licensed dengue vaccine is yet available, several vaccine candidates are under phases of development, including live attenuated virus vaccines, live chimeric virus vaccines, inactivated virus vaccines, subunit vaccines, DNA vaccines and viral-vectored vaccines. Although some vaccine candidates have progressed from animal trials to phase II and III in humans, a number of issues regarding implementation of dengue vaccine in countries like India still need to be addressed. Despite the current limitations, collaborative effects of regulatory bodies like World Health Organization with vaccine manufacturers and policy makers, to facilitate vaccine development and standardize field trials can make a safe and efficacious dengue vaccine a reality in near future.     

Immunity and immunopathology in dengue virus infections.

Dengue virus infections are a serious public health problem in tropical and subtropical areas of the world. Based on epidemiological data, it has been postulated that immune responses to dengue virus contribute to the pathogenesis of severe dengue illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Host immune responses are also important for controlling dengue virus infection. Therefore, dengue virus infections are an interesting model to explore the interactions between viruses and the immune system which result in immunopathology or recovery from infection. In this paper, we review immune responses to dengue viruses with an emphasis on the human T cell responses, and discuss possible roles of these immune responses in the control of dengue virus infection and in the pathogenesis of DHF/DSS.     

Comparison of rapid centrifugation assay with conventional tissue culture method for isolation of dengue 2 virus in C6/36-HT cells

A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results suggest that the rapid centrifugation assay may be useful for detection of dengue virus in clinical specimens.     

Macrophage Activation Syndrome-Associated Markers in Severe Dengue

Hemophagocytosis, a phenomenon of which activated macrophages phagocytosed hematopoietic elements was reportedly observed in severe dengue patients. In the present study, we investigated whether markers of macrophage activation syndrome (MAS) can be used as differential diagnostic markers of severe dengue. Two hundred and eight confirmed dengue patients were recruited for the study. Sandwich ELISA was used to determine serum ferritin, soluble CD163 (sCD163), and soluble CD25 (sCD25) levels. The population of circulating CD163 (mCD163) monocytes was determined using flow cytometry. Receiver operating characteristic (ROC) analysis was plotted to determine the predictive validity of the biomarkers. Serum ferritin and sCD163 were found significantly increased in severe dengue patients compared to dengue fever patients (P = 0.003). A fair area under ROC curves (AUC) at 0.72 with a significant P value of 0.004 was observed for sCD163. sCD25 and mCD163 levels were not significantly different between severe dengue and dengue fever patients. Our findings suggest that in addition to serum ferritin, sCD163 can differentiate severe dengue from that of dengue fever patients. Hence, sCD163 level can be considered for use as a predictive marker for impending severe dengue.     

用逆转录—聚合酶链反应检测海南岛登革热流行区？…

目的 寻找中国海南岛一带登革热疫区，登革病毒潜伏的动物宿主及鉴定其毒株型别。方法 采用１￣４型登革病毒通用引物，用塑转录－聚合酶链反应（ＲＴ－ＰＣＲ）检测海南岛登革热流行区蝙蝠脑细胞，血清和埃及伊蚊的登革病毒ＲＮＡ。结果 检测３５例疫区蝙蝠脑细胞，２０例阳性；检测１８例蝙蝠血清，３例阳性；检测三组埃及伊蚊，１组阳性，３组非流行区者都阴性。用４个登革病毒原型株的单克隆荧光抗体技术检测２０例登革病毒Ｒ     

Imported dengue from 2013 Angola outbreak: Not just serotype 1 was detected

BackgroundAll the reports from Angola’s 2013 dengue outbreak revealed serotype 1. However, previously dengue serotypes 1–4 have been reported in Africa and in 2014 serotype 4 was reported in Angola.ObjectivesTo report dengue serotypes in patients returning from Angola during 2013 outbreak.Study designRetrospective, cross-sectional study. We serotyped the dengue by an in house Polymerase Chain Reaction technique in randomly selected cases.ResultsFrom the 2013 Angola’s dengue outbreak we treated 47 adult patients. None had history of past dengue. A combo kit test for dengue revealed positive NS1 antigen in 39 and IgM antibodies in 8. From 17 randomly patients tested by RNA Real Time-PCR, 11 were positive: 7 for DENV-1, 2 for DENV-2, 1 for DENV-3 (co-infected with DENV-1) and 1 for DENV-4. None had a complicated or fatal evolution.ConclusionUnlike previous reports the 4 serotypes were detected, and this resulted in a different epidemiological situation, raising the risk of future outbreaks of severe dengue.