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Effect of immunosuppression on dengue virus infection in mice.

Mean survival time following intracerebral inoculation of dengue virus was reduced and the titre of the virus in the brain of immunosuppressed mice was markedly increased. A single dose of cyclophosphamide given 24 h after dengue virus i.c. or i.p. substantially reduced the number of antibody forming cells in the spleen. Three doses of dengue virus, each followed by cyclophosphamide 24 h later, produced specific hyporesponsiveness to the dengue virus but not to a heterologous virus (Coxsackie B4), with a reduction in antibody forming cells in the spleen of such animals against dengue virus but not against Coxsackie B4 virus. Adoptive immunity by antiserum was abolished along with increased titres of the virus in the brain of immunosuppressed mice but the protection could be restored by a second dose of antiserum. Pre-treatment of mice with immune or normal spleen cells i.v. or reconstitution of immunosuppressed mice by such cells had no effect. Thus, humoral antibodies play a crucially important role in host defence mechanism in recovery of mice from primary dengue virus infection.

Animals↗

Gene expression in human macrophages infected with dengue virus serotype-2.

Infection by any of the four serotypes of dengue viruses (DEN-1, -2, -3 and -4) may result in either a relatively benign fever, called dengue fever (DF), a fatal disease, such as dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS). Several lines of evidence suggest that soluble immune response mediators may be involved in the severity of dengue infections. For instance, elevated seric levels of IL-8 are a common feature in DHF patients. Because other chemokines, cytokines, adhesion molecules, chemokine and cytokine receptors, as well as cytokine-related molecules may also be involved in dengue virus pathogenesis, we aimed at analysing the gene expression of such molecules in the course of an in vitro DEN-2 infection of human peripheral blood monocyte-derived macrophages, a cell type regarded as a primary target for DEN. Nylon membrane gene arrays containing 375 different human cytokine-related genes were used as a first step to search for differentially expressed genes upon infection. Transcripts for IL-8, IL-1beta, osteopontin, GRO-alpha, -beta and -gamma, I-309, and some other molecules showed to be upregulated upon infection, whereas others such as MIC-1, CD27L and CD30L, were downregulated. Four genes were selected for reverse transcriptase-polymerase chain reaction based gene-expression analysis as a way to partially confirm microarray results. This approach pointed out 25 macrophage-expressed cytokine-related genes that could be relevant in DEN-2 pathogenesis.

Dengue↗

Multiple outbreaks of dengue serotype 2 in north Queensland, 2003/04.

OBJECTIVES: To describe the various investigations and responses to multiple outbreaks of dengue serotype 2 that occurred in north Queensland in 2003/04. METHODS: Details about each case were collated so as to target mosquito-control responses including control of mosquito breeding sites, interior spraying of selected premises, and a novel 'lure and kill' approach using lethal ovitraps. Phylogenetic analyses were undertaken to determine the genetic relatedness of viruses isolated during the outbreaks. RESULTS: Except for a two-month hiatus in mid-2003, the outbreaks continued for 16 months and included approximately 900 confirmed cases, with three severe cases and one death. The available evidence suggests that the mosquito-control measures were effective, but delays in recognising the outbreaks in Cairns and the Torres Strait coupled with intense mosquito breeding contributed to the extensive nature of the outbreaks. Phylogenetic analyses showed that there had been only two major outbreaks, one that spread from Cairns to Townsville, the other from the Torres Strait to Cairns; both were initiated by viraemic travellers from Papua New Guinea. CONCLUSIONS: Phylogenetic analyses were essential in understanding how the outbreaks were related to each other, and in demonstrating that dengue had not become endemic. Further innovative approaches to dengue surveillance and mosquito control in north Queensland are necessary. IMPLICATIONS: Dengue outbreaks have become more frequent and more severe in north Queensland in recent years, raising the possibility that dengue viruses could become endemic in the region leading to outbreaks of dengue haemorrhagic fever.

Aedes↗

[3 dengue fever cases, infected during a group tour to the Philippines].

We report three dengue fever cases, infected during a group tour to the Philippines. A 58-year old male experienced sudden onset of high fever 5 days after returning to Japan, followed by rash and thrombocytopenia. The other 2 cases experienced similar symptoms. Clinically suspected from the travel history, incubation time and the state of dengue fever epidemic in the Philippines, dengue virus infection was confirmed by the laboratory tests. The incidence and geographical distribution of dengue virus infection have greatly increased in recent years. There have been reports of Japanese travelers who visited dengue endemic countries, infected and developed symptoms after returning home. Dengue virus infection should be included in the differential diagnosis of the patients who develop high fever and rash after returning from tropical areas.

Antibodies, Viral↗

Clinical evaluation of a rapid immunochromatographic test for the diagnosis of dengue virus infection.

A rapid immunochromatographic test was compared to the hemagglutination inhibition assay for separate determinations of dengue virus-specific immunoglobulin M (IgM) and IgG levels in paired serum specimens from 92 patients (34 with primary dengue virus infection, 35 with secondary dengue virus infection, and 23 without dengue virus infection). The rapid test showed 99% sensitivity in the diagnosis of dengue virus infection. The majority (30 of 34 [88%]) of patients with primary infection showed positive IgM but negative IgG, while 34 of 35 (97%) patients with secondary infection showed positive IgG with or without IgM. Specificity in nonflavivirus infections was 96% (1 of 23 positive). The rapid test should be a useful aid in rapid diagnosis of dengue virus infection.

Antibodies, Viral↗

Effect of immune status on dengue 2 virus replication in cultured leukocytes from infants and children.

Cord blood leukocytes from neonates with maternal dengue antibody supported dengue 2 virus replication in vitro; those from neonates without maternal antibody did not. Cord bloods of infants born to dengue-immune mothers contained a potent enhancing factor which gradually decayed with age and which was absent from neonates born to nonimmune mothers. Permissiveness of cultures of washed peripheral blood leukocytes from infants with maternal antibody declined steadily with increasing age in parallel with the decay of maternal antibody, and the leukocytes were no longer permissive after 10 to 12 months. The demonstration of a dengue maternal infection-enhancing factor in human cord blood from dengue-immune mothers supports the hypothesis that severe primary dengue hemorrhagic fever with shock seen in Bangkok infants is related to maternal immune status.

Age Factors↗

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.

DNA Primers↗

Potential application of nonstructural protein NS1 serotype-specific immunoglobulin G enzyme-linked immunosorbent assay in the seroepidemiologic study of dengue virus infection: correlation of results with those of the plaque reduction neutralization test.

An NS1 serotype-specific indirect enzyme-linked immunosorbent assay (ELISA) was developed to differentiate primary and secondary dengue virus infections and serotypes of primary dengue virus infection. For this report, we carried out retrospective seroepidemiologic studies on serum samples collected from residents of Liuchiu Hsiang, Pingtung County, an isolated island in southern Taiwan during 1997-1998. The results demonstrated that good correlation existed between dengue virus NS1 serotype-specific immunoglobulin G (IgG) ELISA and dengue virus plaque reduction neutralization test (PRNT). Our data suggested that NS1 serotype-specific IgG ELISA could replace PRNT for seroepidemiologic studies to differentiate Japanese encephalitis and dengue virus infections and for dengue virus serotyping.

Adolescent↗

Castanospermine, a potent inhibitor of dengue virus infection in vitro and in vivo.

Previous studies have suggested that alpha-glucosidase inhibitors such as castanospermine and deoxynojirimycin inhibit dengue virus type 1 infection by disrupting the folding of the structural proteins prM and E, a step crucial to viral secretion. We extend these studies by evaluating the inhibitory activity of castanospermine against a panel of clinically important flaviviruses including all four serotypes of dengue virus, yellow fever virus, and West Nile virus. Using in vitro assays we demonstrated that infections by all serotypes of dengue virus were inhibited by castanospermine. In contrast, yellow fever virus and West Nile virus were partially and almost completely resistant to the effects of the drug, respectively. Castanospermine inhibited dengue virus infection at the level of secretion and infectivity of viral particles. Importantly, castanospermine prevented mortality in a mouse model of dengue virus infection, with doses of 10, 50, and 250 mg/kg of body weight per day being highly effective at promoting survival (P < or = 0.0001). Correspondingly, castanospermine had no adverse or protective effect on West Nile virus mortality in an analogous mouse model. Overall, our data suggest that castanospermine has a strong antiviral effect on dengue virus infection and warrants further development as a possible treatment in humans.

Animals↗

Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses.

BACKGROUND: In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses. RESULTS: Convalescent sera from 69 individuals who were known to have had dengue or Japanese encephalitis virus infection were tested by western blotting against dengue, Japanese encephalitis and West Nile virus antigens. We determined that individuals who had been infected with dengue viruses had IgG responses against the premembrane protein of dengue viruses but not Japanese encephalitis, whereas individuals who had been infected with Japanese encephalitis had IgG specific for the premembrane protein of Japanese encephalitis virus but not the dengue viruses. None reacted with the premembrane protein of West Nile virus. Using the Pearson Chi Square test, it was determined that the difference between the two groups was highly significant with a p value of <0.001. CONCLUSION: The use of flavivirus premembrane protein in seroepidemiological studies will be useful in determining what flaviviruses have circulated in a community.

Aedes↗

Epidemic dengue 4 in the Yucatán, México, 1984.

An outbreak of dengue 4 occurred in the Yucatán, México in 1984. During the course of the outbreak, 538 of 5486 reported cases of dengue-like illness were studied; 200 were confirmed as dengue serologically and/or virologically. Dengue 4 virus was isolated from 34 patients and dengue 1 from one. Severe haemorrhagic symptoms were observed in 9 laboratory confirmed patients, including four deaths. Thus, the outbreak in Yucatán is the second dengue epidemic in the Americas after the Cuban epidemic in 1981 in which a number of patients suffered from haemorrhagic complications. It was notable that 5 of 9 hospitalized, severe cases were young adults and that only one met the WHO criteria of DHF, in contrast to primary pediatric nature of DHF in Southeast Asia. In this paper we describe clinical, serologic, and virologic studies conducted during the outbreak.

Adolescent↗

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen.

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

Animals↗

[Polymerase chain reaction for rapid detection and serotyping of dengue virus in clinical samples].

This study describes the benefits of using reverse transcriptase polymerase chain reaction (RT-PCR) for the rapid detection and typing of dengue virus in clinical samples. Twenty-seven serum specimens from patients with dengue fever and dengue hemorrhagic fever in Colombia, Nicaragua, and Panama were directly subjected to RT-PCR for the detection of dengue virus. The resulting double-stranded DNA product was typed by a second round of PCR amplification (nested PCR) with type-specific primers, viral culture/indirect immunofluorescence (IIF), and enzyme-linked electroimmunoassay for IgM anti-dengue antibodies. The amplified virus genome was detected and typed within 8 hours. Nested RT-PCR, using viral culture and IIF as the gold standard, showed 100% sensitivity; 78% specificity; 69% positive predictive value, and 100% negative predictive value. It is noteworthy that two of the specimens whose results were positive with nested RT-PCR and negative with viral culture showed specific IgM antibodies. The results of the RT-PCR were in close agreement with those obtained through viral culture. This suggests PCR can greatly facilitate the rapid and early diagnosis of dengue infection.

Colombia↗

A case of dengue from Pune, India.

Dengue is an acute infectious disease, caused by a single stranded ribonucleic acid (RNA) virus of the family of Flaviviridae, genus Flavivirus, transmitted by Aedes mosquitos, the most important vectors being Aedes aegypti and Aedes albopictus. There are four serotypes, DEN-1, DEN-2, DEN-3, DEN-4 that are nearly human specific. The geographical distribution of dengue is pantropical, except for Madagascar and some African regions. It is endemo-epidemic in tropical and subtropical countries included between 25 degrees north latitude and 25 degrees south latitude, particularly in southeast Asia and epidemic in the Caribbean, West-Africa, tropical American and Pacific Islands. Further spread will depend on the invasion of Aedes aegypti into new areas in South America. Maintenance of dengue is supported by an increasing demography, uncontrolled urbanization and climatic conditions favorable to the vectors. Spread of dengue is primarily caused by modern transportation, especially aircraft. No vaccine and no specific treatment exist so that mosquito control is the only way to reduce the incidence of dengue around the world. Dengue is, obviously, an important risk for travelers going to endemic areas.

Adult↗

Molecular and epidemiologic analysis of dengue virus isolates from Somalia.

Nucleotide sequence analysis was performed on 14 dengue virus isolates (13 dengue-2 viruses and 1 dengue-3 virus) recovered from febrile soldiers in Somalia in 1993. The dengue-2 viruses were most closely related to dengue-2 virus recovered in Somalia in 1984. However, differences in nucleotide sequence (0.35% to 1.35%) were evident among the 1993 isolates. These differences were closely associated with the geographic location of the infection as well as with different times of infection at the same location. Genetic difference between strains was not associated with differences in clinical features. Molecular analysis of dengue viruses is a useful adjunct to epidemiologic investigation of their distribution over distance and time.

Adult↗

T cell responses in dengue hemorrhagic fever: are cross-reactive T cells suboptimal?

Dengue virus infection poses a growing public health and economic burden in a number of tropical and subtropical countries. Dengue circulates as a number of quasispecies, which can be divided by serology into four groups or serotypes. An interesting feature of Dengue, recognized over five decades ago, is that most severe cases that show hemorrhagic fever are not suffering from a primary infection. Instead, they are reinfected with a virus of different serotype. This observation poses considerable problems in vaccine design, and it is therefore imperative to gain a full understanding of the mechanisms underlying this immunological enhancement of disease. In this study, we examined a T cell epitope restricted by HLA-A*24, a major MHC class I allele, in Southeast Asia in a cohort of children admitted to a hospital with acute Dengue infection. The cytokine profiles and the degranulation capacity of T cells generated to this epitope are defined and compared across different viral serotypes. Cross-reactive Dengue-specific T cells seem to show suboptimal degranulation but high cytokine production, which may contribute to the development of the vascular leak characteristic of Dengue hemorrhagic fever.

Amino Acid Sequence↗

Etiologies of the experimental dengues of Siler and Simmons.

Four participants in the American Army Philippine dengue (d) studies of Siler (1924-25) and five subjects from the studies of Simmons (1929-30) were bled 42 to 48 years after experimental infection. Sera were studied by hemagglutination-inhibition, complement-fixation and plaque reduction neutralization (PRNT) tests for antibodies to the known viral causes of the dengue syndrome in Asia and to St. Louis encephalitis virus. PRNT tests were done with and without fresh normal human serum (accessory factor). Serum from one of four Siler study volunteers had unequivocal evidence of only a previous d4 infection; the remainder had broadly reactive group B antibodies, including d4, suggesting past infection with two or more different dengue viruses. Sera from four of the Simmons study group had high-titered PRNT antibodies to d1 and the remaining serum had d1 antibody when accessory factor was added. In three sera there was monotypic PRNT antibody to d1. Acessory factor increased PRNT titers, particularly to d1 virus in the Simmons group. Only sera with antibody patterns suggesting past infections with two or more dengue viruses had high levels of SLE PRNT antibody. It is concluded that d4 was transmitted in the 1924 to 1925 and d1 in the 1929 to 1930 experiments. Notable differences in clinical features of dengue infections in the two studies suggest the possibility of the existence of an unique spectrum of responses to infection with different dengue virus type.

Adult↗

Clinical and laboratory observations on patients with primary and secondary dengue type 1 infections with hemorrhagic manifestations in Fiji.

An extensive epidemic of dengue type 1 infection with a high incidence of hemorrhagic manifestations occurred on the island of Viti Levu, Fiji beginning early in 1975. Previous dengue outbreaks in this population were such that in 1975 two types of dengue patients were observed, one group of patients experienced primary dengue type 1 infection, whereas another experienced their dengue type 1 infection approximately 4 years after a dengue type 2 infection. Clinical and laboratory findings for the two forms of infection were assessed in patients hospitalized for their disease, usually with hemorrhage. With the exception of virologic and serologic findings, no important differences between these two groups were noted with respect to incidence and nature of hemorrhage and other clinical and laboratory findings. Both types of infection sometimes were associated with thrombocytopenia and low serum levels of the C3 component of complement. Virus was more easily isolated from the primary infection patients and the magnitude of their viremia also was higher. No significant differences were noted in a comparison of the ease of virus isolation from serum or plasma.

Adolescent↗