Showing papers on "Dengue virus published in 1992"

Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

Abstract: We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

Abstract: The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.

Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5.

Abstract: Processing of dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5 could be mediated by the catalytically active NS3 protease domain and NS2B in trans at the dibasic sites NS3-NS4A and NS4B-NS5. Subcellular localization of the unprocessed precursor NS3-NS4A-NS4B-NS5 showed that it was confined to a distinct subcellular organelle in the cytoplasm, which was distinct from the distribution of the mature NS5.

Dengue haemorrhagic fever in children in Delhi.

Abstract: An epidemic of dengue haemorrhagic fever occurred in Delhi during 1988. A total of 21 paediatric patients with dengue haemorrhagic fever/dengue shock syndrome were evaluated from September to November 1988. All the patients had fever, restlessness, ecchymotic spots and ascites. Pleural effusion occurred in 19 patients (90%), and 18 (86%) exhibited each of the following: vomiting, thrombocytopenia, and haemoconcentration. Hepatomegaly was observed in 15 patients (71%) and splenomegaly in three (14%). Titres of haemagglutination inhibition (HI) antibodies against dengue virus type 2 were raised in all the 15 cases from whom sera were collected during the acute stage. Convalescent sera from five patients had increased titres of HI antibodies to dengue virus type 2. The remaining 10 cases exhibited raised IgM antibody levels against dengue virus type 2. The fatality rate for serologically proven cases was 13% (2 of 15 patients), while for all patients (including those diagnosed clinically (6) and serologically (15)) it was 33.3% (7 of 21). Patients who survived had no sequelae, except one who had transient hypertension that lasted for two weeks.

Chimeric and/or growth-restricted flaviviruses

Abstract: The invention includes a chimeric virus for use in a vaccine preparation having a genome comprising nucleic acid sequences encoding at least one structural protein from one flavivirus and nucleic acid sequences encoding nonstructural protein from another flavivirus. The genome preferably includes mutations within the viral genome that reduce virus virulence and in a particularly preferred embodiment these vaccines are directed to flaviviruses such as dengue virus, tick-borne encephalitis virus and Japanese encephalitis virus.

First reported outbreak of classical dengue fever at 1,700 meters above sea level in Guerrero State, Mexico, June 1988.

Abstract: An outbreak of classical dengue fever occurred from March to August 1988 in the city of Taxco, Guerrero State, Mexico. Taxco is at an elevation of 1,700 meters above sea level, and this study represents the highest altitude at which an outbreak of dengue has been documented. An investigation was conducted to obtain serologic confirmation of dengue infection, determine the extent of the outbreak, and identify risk factors for dengue illness. Toxorhynchites cell lines were used for viral isolation, and hemagglutination inhibition was used to measure anti-dengue antibody titers. The case definition used in the investigation was any person with fever, headache, myalgias, and arthralgias, or rash or retroocular pain. Dengue virus type 1 was isolated from five acute cases. Of 1,686 persons living in the affected area, 42% (715) met the case definition. Large (200-liter) water containers were significantly associated with infection (relative risk = 1.7, 95% confidence interval 1.5-1.9). The effect of altitude on epidemic transmission is most likely modulated by seasonal temperatures. The epidemiologic and serologic confirmation of a dengue outbreak at 1,700 meters above sea level represents the capability of Aedes aegypti to adapt to new environments, and the potential for epidemic spread in cities at comparable altitudes or higher.

Correlation of E protein binding with cell susceptibility to dengue 4 virus infection

Abstract: Supernatant culture fluids from dengue virus type 4 (DEN-4)-infected cultures of monkey kidney Vero cells and Aedes albopictus C6/36 cells contained the virion structural proteins; secreted NS1 was found only in supernatants from infected Vero cells. Using supernatant culture fluids from [35S]methionine-labelled, virus-infected Vero and C6/36 cells, binding of radiolabelled viral proteins was examined with various cell lines varying in susceptibility to DEN-4 infection. Binding of viral E protein was observed with the highly infectible Vero and LLC-MK2 cell lines, whereas a very small degree of binding was seen with four other cell lines (mouse fibroblast L929, bovine kidney MDBK, human hepatoma Hep G2 and primary human endothelial cells) which are less susceptible to DEN-4 infection. The results suggest that cell susceptibility to DEN-4 may be determined largely at the stage of virus binding, i.e. by the presence of a cell receptor capable of binding viral E protein.

Dengue virus infection of human skin fibroblasts in vitro production of IFN-beta, IL-6 and GM-CSF.

Abstract: Dengue virus is transmitted to humans by the bite of infected mosquitos. In our efforts to understand the pathogenesis of dengue virus infection, we examined whether skin fibroblasts can be infected in vitro with dengue viruses. Fibroblasts could be infected with dengue viruses, yellow fever virus and West Nile virus. Dengue virus antigen-positive cells were detected as early as 4h and the percentage of dengue virus antigen-positive cells reached maximum levels by 24h after infection. High titers of infectious dengue virus were also detected in the culture supernatants at 20h after infection. Dengue virus-infected fibroblasts produced interferon-β (IFN-β), and the IFN-β protected uninfected fibroblasts from dengue virus infection. Dengue virus-infected fibroblasts also produced interleukin 6 (IL-6) and granulocyte macrophage colony stimulation factor (GM-CSF).

The Predisposing and Protective Factors against Dengue Virus Transmission by Mosquito Vector

Abstract: An outbreak of dengue fever occurred in Taiwan between 1987 and 1988. The highest attack rate among adults was estimated at 5.6% in the city of Kao-hsiung. A case-control study was carried out to determine the risks of contracting dengue infection and to identify protective factors against the infection. One hundred dengue patients of the authors' hospital who were diagnosed by virologic or serologic tests constituted the case group. Each dengue patient was matched to a control patient of the same age and sex who had been diagnosed as suffering from a non-vector-borne disease on the same day as the dengue patient. Of the household protective measures against dengue infection prior to the occurrence of illness, the adjusted odds ratio, estimated by stratified analysis, was lower for people who lived in screened houses (odds ratio = 0.58, 95% confidence interval 0.36-0.92) as compared with inhabitants of unscreened houses. The odds ratio was as low as 0.18 (95% confidence interval 0.06-0.56) for people whose homes were fully screened with door screens opening outwardly. Patients who lived near markets and/or open sewers or ditches were running a risk of dengue infection 1.8 (95% confidence interval 1.3-2.4) times higher than those who lived elsewhere. To control dengue outbreaks, the authors recommend that special attention should be devoted to the reduction of outdoor vector sources. Full screening, especially outwardly opening screen doors, seems to be an individual's best protection against dengue fever.

Envelope protein sequences of dengue virus isolates TH-36 and TH-Sman, and identification of a type-specific genetic marker for dengue and tick-borne flaviviruses

Abstract: Complementary DNAs were synthesized from the envelope protein genes of two isolates of dengue virus (TH-36 and TH-Sman, previously suggested as possible dengue virus type 5 and dengue virus type 6 respectively) and amplified by the polymerase chain reaction using sense and antisense primers designed from conserved dengue virus gene sequences. The amplified cDNA clones were sequenced in both directions by double-stranded dideoxynucleotide sequencing. Alignment with published dengue virus sequences enabled us to assign these viruses accurately to classified serotypes, confirming that TH-36 and TH-Sman are strains of dengue virus type 2 and dengue virus type 1 respectively. Amino acid changes between the proteins encoded by these two isolates and strains of their respective serotypes may account for the significant antigenic differences observed during previous serological typing of these viruses. Moreover, sequence alignment of flavivirus envelope proteins revealed a hypervariable region, within which members of the dengue and tick-borne virus antigenic complexes show unique peptide sequences. This type-specific hypervariable domain may be useful as a genetic marker for typing dengue and tick-borne flaviviruses.

Estudo sobre o diagnóstico laboratorial e sintomas do dengue, durante epidemia ocorrida na região de Ribeirão Preto, SP, Brasil

Abstract: A dengue type 1 outbreak started in the Ribeirao Preto Region, North of Sao Paulo State, Brazil, in November of 1990. About 3500 dengue cases were confirmed by blood tests until February of 1991. The Virus Research Unit of The Faculty of Medicine of Ribeirao Preto - Sao Paulo State University, studied 502 dengue suspect cases. The Serologic diagnosis of dengue type 1 was confirmed by haemmaglutination inhibition test (HAI) in 19% of the cases. Diagnosis was done later by using an enzyme immuno assay on infected cultured cells (EIA-ICC) which discriminated IgG and IgM dengue, antibodies. EIA-ICC was less sensitive (89%) but more effective than HAI. EIA-ICC is a simple technique. It dispenses a second serum sample for diagnosis and it can be completed in about 5 hours. Dengue virus was isolated from the blood of 21 patients by innoculation in culture of mosquito C6/36 cells. The isolated virus were identified by indirect immunofluorescent test, by using an antisera pool to the flavivirus family and dengue type specific monoclonal antibodies. The dengue most frequent symptoms in 71 patients were observed: fever (90%), myalgias (57%) and arthralgias(41%)

Failure of dengue viruses to replicate in Culex quinquefasciatus (Diptera: Culicidae).

Abstract: Culex quinquefasciatus (Say) and Aedes aegypti (L.) were parenterally infected with dengue viruses and virus replication was monitored at intervals after infection in each species. Dengue viruses replicated rapidly in Ae. aegypti, reaching a peak titer of 106–107 mosquito infectious dose 50 (MID50) per mosquito. In Cx. quinquefasciatus, however, dengue virus replication did not occur. We conclude that this mosquito species is refractory to infection with dengue viruses and, therefore, does not serve as a vector in nature.

Brazilian dengue virus type 1 replication in mosquito cell cultures

Abstract: The development of dengue viruses type 1 obtained from acute human sera and inoculated into mosquito cell cultures, was observed by standard transmission electron microscopy and cytochemical staining. It follows the trans-type mechanism already established of other dengue types. Direct passage of single virus particles across the cell membrane seems to be a pathway of entry and exit in dengue-1 infected cells. The nature of numerous electron translucent vesicles and tubules, produced simultaneously during virus replication inside the rough endoplasmic reticulum, was analyzed by cytochemical tests. The largest amount of virus particles was produced inside cell syncytia.

Processing of dengue virus type 2 structural proteins containing deletions in hydrophobic domains.

Abstract: The 5′ end of the genome of the dengue virus type 2 encoding the structural proteins was expressed using recombinant vaccinia virus. Three additional recombinants derived by deletion of selected dengue sequences within the parental construct were also expressed. They were designed to assess the role of hydrophobic domains in the processing of the viral polyprotein in intact cells. The first construct contained a deletion of nucleotides encoding most of the C protein; nucleotides encoding the hydrophobic domain at the carboxy terminus were retained. The second and third constructs contained smaller deletions of 72 bp and 129 bp encoding hydrophobic domains at the carboxy termini of C and prM respectively. Indirect immunofluorescence and radioimmunoprecipitation were used to detect prM and E in cells infected with recombinant viruses. The results showed that deletion of 90% of C had no apparent effect on the processing of prM and E, and that the signal sequence for E at the carboxy terminus of prM was active in the absence of the upstream signal sequence for prM at the carboxy terminus of C. Deletion of the hydrophobic sequences preceding the amino terminus of E prevented cleavage at the prM-E junction. These results obtained using infected cells were consistent with the published findings for the translation of flavivirus RNA in vitro, and indicated the importance of membrane association in the cleavage of structural proteins from the flavivirus polyprotein. In addition, cells infected with the recombinant virus containing the large deletion in the C coding region released the E glycoprotein into the culture medium.

Human antibodies to dengue and yellow fever do not react in diagnostic assays for hepatitis C virus.

Abstract: Hepatitis C virus (HCV) is a recently described causative agent of the great majority of post-transfusion non A-non B hepatitis and is classified within the Flaviviridae family. Due to a high prevalence of anti-HCV and other flaviviruses circulating in Brazil, such as dengue and yellow fever, we investigated the possibility of serological cross-reactivity between these viruses. Different panels of human sera positive for dengue type 1 (9 cases) and type 2 (7 cases) from 6 patients naturally infected with yellow fever and from 94 adults vaccinated against the 17D strain of yellow fever were tested against HCV antigens used in diagnostic assays. Two enzyme immunoassay systems were tested: one, an in-house test using recombinant antigens from core, NS3 and NS5 regions of the HCV genome (Research Foundation for Microbial Disease of Osaka University, Japan); and another, using synthetic peptides representing immunodominant epitopes of structural core and non-structural NS4 and NS5 HCV regions (INNOTEST HCV Ab, Innogenetics, Belgium). A line immunoassay (INNO-LIA HCV Ab, Innogenetics, Belgium) was used as a confirmatory test. In this, HCV antigens are coated as discrete lines on a nylon strip with plastic backing. Besides 4 control lines on each strip, a total of 6 HCV lines are present: line A consists of several NS4 epitopes, line B consists of several NS5 epitopes and lines C-F contain several core epitopes. This test not only confirms but differentiates antibodies to hepatitis C virus. No positive results were detected with these tests, indicating that hepatitis C infection can be evaluated by current assays in regions where flaviviruses are endemic.

Experimental studies on the susceptibility of Aedes vittatus to dengue viruses.

Abstract: Ae. vittatus mosquitoes were infected by oral route and by intrathoracic inoculation with dengue (DEN) viruses and tested for the presence of dengue virus antigen in their head squashes and salivary glands by indirect immunofluorescence. The results indicate that this species was susceptible to all four types of DEN viruses and supported the growth of DEN-2 virus.

[Use of a dengue anti-complex monoclonal antibody in viral purification].

Abstract: The purification of different serotypes of dengue virus from a monoclonal antibody which recognizes an epitope present in the four serotypes bond to a Sepharose 4B matrix activated with cyanogen bromide, is reported. Results evidence that the method employed makes quicker the process of purification with a high degree of purity.

Infection with Dengue virus.

Abstract: We report a male Caucasian, with a Dengue virus infection imported from Thailand to The Netherlands General characteristics of the disease are presented and the supposed pathogenetic mechanisms of the disease are discussed

An enzyme-linked immunosorbent assay using a chaotropic agent (sodium thiocyanate) for serotype specific reaction between crude dengue viral antigen and anti-dengue mouse antibody.

Abstract: An enzyme-linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti-dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement-fixing antigen (SCF) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted serotype specifically to anti-dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti-type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti-type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.