A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.
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ABSTRACT:
SUBMITTER: Diaz-Badillo A
PROVIDER: S-EPMC4063023 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
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A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.
Díaz-Badillo Alvaro A Muñoz María de Lourdes Mde L Perez-Ramirez Gerardo G Altuzar Victor V Burgueño Juan J Mendoza-Alvarez Julio G JG Martínez-Muñoz Jorge P JP Cisneros Alejandro A Navarrete-Espinosa Joel J Sanchez-Sinencio Feliciano F
Sensors (Basel, Switzerland) 20140425 5
Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridise ...[more]
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Project description:Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. Background Dengue is a mosquito-borne viral infection causing a major public health problem globally. Dengue virus (DENV) is the causative agent of dengue fever (DF) and dengue hemorrhagic fever (DHF) and includes four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). DENV-2 and DENV-3 have been associated with severe dengue disease, consequently, laboratory testing for DENV is needed to confirm the diagnosis of DENV infection, serotype and to differentiate dengue from other febrile tropical illnesses. In addition, surveillance of mosquitoes infected with DENV is needed to monitor the infection rates within vector mosquito populations harboring specific serotype to provide an early warning sign to predict epidemics. Results In this work we have applied microarray analysis to simultaneously determine the serotype of multiple RNA samples from human or mosquitoes. The proposed microarray method can be used for i) rapid and reliable dengue diagnosis; ii) serotyping and iii) surveillance of mosquitoes infected with dengue. These microarrays were useful to confirm the presence of DENV-2 in 94 serum samples, DENV-3 in three samples from Juchitan, Oaxaca and one case from Juchitan, Oaxaca contained DENV-2 and -3. Moreover by using these microarrays we also determined DENV in pools of gravid females mosquitoes collected in several sites of nineteen Mexican states in 2005. Mosquito pools from 31 cities in the states of Yucatan, Campeche, Tabasco, Chiapas, Veracruz, Oaxaca, Guerrero, Tamaulipas and Colima were infected with DENV-2, six cities in Yucatán, Tabasco, Morelos, Tamaulipas, Colima, and Nayarit with DENV-1, three from Tabasco, Veracruz and Oaxaca with DENV 3 and two with two serotypes simultaneously (Ciudad Mante with DENV-1 and DENV-2, and Tavela with DENV-2 and DENV-3). Conclusion Here we show the success of applying microarrays assay to provide a consistently robust qualitative detection of dengue serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) in serum samples from patients or in pools of gravid female mosquitoes collected in the field of nineteen Mexican states. Interestingly, we did not detect any mosquito or serum sample containing DENV-4.
2015-11-30 | GSE26943 | GEO
Project description:BACKGROUNDDisease due to dengue viruses is a growing global health threat, causing 100-400 million cases annually. An ideal dengue vaccine should demonstrate durable protection against all 4 serotypes in phase III efficacy trials, however the lack of circulating serotypes may lead to incomplete efficacy data. Controlled human infection models help downselect vaccine candidates and supply critical data to supplement efficacy trials. We evaluated the efficacy of a leading live-attenuated tetravalent dengue vaccine candidate, TV005, against infection with a newly established dengue serotype 3 or an established serotype 2 challenge virus.METHODSTwo randomized, controlled clinical trials were performed. In study 1, a total of 42 participants received TV005 or placebo (n = 21 each), and 6 months later, all were challenged with dengue 2 virus (rDEN2Δ30) at a dose of 103 PFU. In study 2, a total of 23 participants received TV005 and 20 received placebo, and 6 months later, all were challenged with 104 PFU dengue 3 virus (rDEN3Δ30). The study participants were closely monitored for safety, viremia, and immunologic responses. Infection, measured by post-challenge viremia, and the occurrence of rash and neutropenia were the primary endpoints. Secondary endpoints included safety, immunologic, and virologic profiles following vaccination with TV005 and subsequent challenge with the rDEN2Δ30 or rDEN3Δ30 strain.RESULTSTV005 was well tolerated and protected all vaccinated volunteers from viremia with DENV2 or DENV3 (none infected in either group). Placebo recipients had post-challenge viremia (100% in study 1, 85% in study 2), and all experienced rash following challenge with either serotype.CONCLUSIONSTV005 is a leading tetravalent dengue vaccine candidate that fully protected against infection with DENV2 and DENV3 in an established controlled human infection model.TRIAL REGISTRATIONClinicalTrials.gov NCT02317900 and NCT02873260.FUNDINGIntramural Research Program, NIH (contract HHSN272200900010C).
| S-EPMC10836801 | biostudies-literature
Project description:To determine the causes of viral meningitis, we analyzed 22 cerebrospinal fluid samples collected during the 2014-2015 dengue epidemics in Brazil. We identified 3 serotypes of dengue virus (DENV-1, -2, and -3), as well as co-infection with 2 or 3 serotypes. We also detected the Asian II genotype of DENV-2.
| S-EPMC5176234 | biostudies-literature
Project description:IntroductionDengue virus (DENV) infection is currently a major cause of morbidity and mortality in the world; it has become more common and virulent over the past half-century and has gained much attention. Thus, this review compared the percentage of severe cases of both primary and secondary infections with different serotypes of dengue virus.MethodsData related to the number of cases involving dengue fever (DF), dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) or severe dengue infections caused by different serotypes of dengue virus were obtained by using the SCOPUS, the PUBMED and the OVID search engines with the keywords "(dengue* OR dengue virus*) AND (severe dengue* OR severity of illness index* OR severity* OR DF* OR DHF* OR DSS*) AND (serotypes* OR serogroup*)", according to the MESH terms suggested by PUBMED and OVID.ResultsApproximately 31 studies encompassing 15,741 cases reporting on the dengue serotypes together with their severity were obtained, and meta-analysis was carried out to analyze the data. This study found that DENV-3 from the Southeast Asia (SEA) region displayed the greatest percentage of severe cases in primary infection (95% confidence interval (CI), 31.22-53.67, 9 studies, n = 598, I2 = 71.53%), whereas DENV-2, DENV-3, and DENV-4 from the SEA region, as well as DENV-2 and DENV-3 from non-SEA regions, exhibited the greatest percentage of severe cases in secondary infection (95% CI, 11.64-80.89, 4-14 studies, n = 668-3,149, I2 = 14.77-96.20%). Moreover, DENV-2 and DENV-4 from the SEA region had been found to be more highly associated with dengue shock syndrome (DSS) (95% CI, 10.47-40.24, 5-8 studies, n = 642-2,530, I2 = 76.93-97.70%), while DENV-3 and DENV-4 from the SEA region were found to be more highly associated with dengue hemorrhagic fever (DHF) (95% CI, 31.86-54.58, 9 studies, n = 674-2,278, I2 = 55.74-88.47%), according to the 1997 WHO dengue classification. Finally, DENV-2 and DENV-4 from the SEA region were discovered to be more highly associated with secondary infection compared to other serotypes (95% CI, 72.01-96.32, 9-12 studies, n = 671-2,863, I2 = 25.01-96.75%).ConclusionThis study provides evidence that the presence of certain serotypes, including primary infection with DENV-3 from the SEA region and secondary infection with DENV-2, DENV-3, and DENV-4 also from the SEA region, as well as DENV-2 and DENV-3 from non SEA regions, increased the risk of severe dengue infections. Thus, these serotypes are worthy of special consideration when making clinical predictions upon the severity of the infection.Systematic review registrationPROSPERO CRD42015026093 (http://www.crd.york.ac.uk/PROSPERO).
| S-EPMC4877104 | biostudies-literature
Project description:The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.
| S-EPMC11003990 | biostudies-literature
Project description:Dengue fever, caused by infections with the dengue virus (DENV), affects nearly 400 million people globally every year. Early diagnosis and management can reduce the morbidity and mortality rates of severe forms of dengue disease as well as decrease the risk of wider outbreaks. Although the early diagnosis of dengue can be achieved using a number of commercial NS1 detection kits, none of these can differentiate among the four dengue virus serotypes. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) for the detection of dengue virus (DENV) NS1 by pairing a serotype-cross-reactive monoclonal antibody (MAb) with one of four serotype-specific MAbs in order to facilitate the rapid detection of NS1 antigens and the simultaneous differentiation of DENV serotypes. A total of 146 serum samples obtained from patients suspected to be in the acute phase of DENV infection were used to evaluate the clinical application of our novel test for the detection and serotyping of DENV. The overall sensitivity rate of our test was 84.85%, and the sensitivity rates for serotyping were as follows: 88.2% (15/17) for DENV serotype 1 (DENV1), 94.7% (18/19) for DENV2, 75% (12/16) for DENV3, and 66.6% (6/9) for DENV4. Moreover, there was no cross-reactivity among serotypes, and no cross-reactivity was observed in sera from nondengue patients. Thus, our test not only enables the rapid detection of the dengue virus but also can distinguish among the specific serotypes during the early stages of infection. These results indicate that our ELISA for DENV NS1 is a convenient tool that may help elucidate the epidemiology of DENV outbreaks and facilitate the clinical management of DENV infections.
| S-EPMC6595446 | biostudies-literature
Project description:Dengue virus (DENV) infections are vectored by mosquitoes and constitute one of the most prevalent infectious diseases in many parts of the world, affecting millions of people annually. Current treatments for DENV infections are nonspecific and largely ineffective. In this study, we describe the adaptation of a high-content cell-based assay for screening against DENV-infected cells to identify inhibitors and modulators of DENV infection. Using this high-content approach, we monitored the inhibition of test compounds on DENV protein production by means of immunofluorescence staining of DENV glycoprotein envelope, simultaneously evaluating cytotoxicity in HEK293 cells. The adapted 384-well microtiter-based assay was validated using a small panel of compounds previously reported as having inhibitory activity against DENV infections of cell cultures, including compounds with antiviral activity (ribavirin), inhibitors of cellular signaling pathways (U0126), and polysaccharides that are presumed to interfere with virus attachment (carrageenan). A screen was performed against a collection of 5,632 well-characterized bioactives, including U.S. Food and Drug Administration-approved drugs. Assay control statistics show an average Z' of 0.63, indicative of a robust assay in this cell-based format. Using a threshold of >80% DENV inhibition with <20% cellular cytotoxicity, 79 compounds were initially scored as positive hits. A follow-up screen confirmed 73 compounds with IC₅₀ potencies ranging from 60 nM to 9 μM and yielding a hit rate of 1.3%. Over half of the confirmed hits are known to target transporters, receptors, and protein kinases, providing potential opportunity for drug repurposing to treat DENV infections. In summary, this assay offers the opportunity to screen libraries of chemical compounds, in an effort to identify and develop novel drug candidates against DENV infections.
| S-EPMC2962577 | biostudies-literature
Project description:BackgroundThe search for the dengue virus receptor has generated many candidates often identified only by molecular mass. The wide host range of the viruses in vitro combined with multiple approaches to identifying the receptor(s) has led to the notion that many receptors or attachment proteins may be involved and that the different dengue virus serotypes may utilize different receptors on the same cells as well as on different cell types.ResultsIn this study we used sequential extraction of PS Clone D cell monolayers with the detergent beta-octylglucopyranoside followed by sodium deoxycholate to prepare a cell membrane-rich fraction. We then used 2 dimensional (2D) gel electrophoresis to separate the membrane proteins and applied a modified virus overlay protein binding assay (VOPBA) to show that dengue virus serotypes 1, 2 and 3 all interact with the 37 kDa/67 kDa laminin receptor (LAMR1), a common non-integrin surface protein on many cell types.ConclusionAt least 3 of the 4 dengue serotypes interact with the 37 kDa/67 kDa laminin receptor, LAMR1, which may be a common player in dengue virus-cell surface interaction.
| S-EPMC1079963 | biostudies-literature
Project description:Dengue virus infections can cause hemorrhagic fever, shock, encephalitis, and even death. Worldwide, approximately 2.5 billion people live in dengue-infested regions with about 100 million new cases each year, although many of these infections are believed to be silent. There are four antigenically distinct serotypes of dengue virus; thus, immunity from one serotype will not cross-protect from infection with the other three. The difficulties that hamper vaccine development include requirements of the natural conformation of the envelope glycoprotein to induce neutralizing immune responses and the necessity of presenting antigens of all four serotypes. Currently, the only way to meet these requirements is to use a mixture of four serotypes of live attenuated dengue viruses, but safety remains a major problem. In this study, we have developed the basis for a tetravalent dengue vaccine using a novel complex adenovirus platform that is capable of expressing multiple antigens de novo. This dengue vaccine is constructed as a pair of vectors that each expresses the premembrane and envelope genes of two different dengue virus serotypes. Upon vaccination, the vaccine expressed high levels of the dengue virus antigens in cells to mimic a natural infection and induced both humoral and cellular immune responses against multiple serotypes of dengue virus in an animal model. Further analyses show the humoral responses were indeed neutralizing against all four serotypes. Our studies demonstrate the concept of mimicking infections to induce immune responses by synthesizing dengue virus membrane antigens de novo and the feasibility of developing an effective tetravalent dengue vaccine by vector-mediated expression of glycoproteins of the four serotypes.
| S-EPMC1797786 | biostudies-literature
Project description:The Dengue virus (DENV) constitutes a major vector borne virus disease worldwide. Prediction of the DENV spread dynamics, prevalence and infection rates are crucial elements to guide the public health services effort towards meaningful actions. The existence of four DENV serotypes further complicates the virus proliferation forecast. The different serotypes have varying clinical impacts, and the symptomatology of the infection is dependent on the infection history of the patient. Therefore, changes in the prevalent DENV serotype found in one location have a profound impact on the regional public health. The prediction of the spread and intensity of infection of the individual DENV serotypes in specific locations would allow the authorities to plan local pesticide spray to control the vector as well as the purchase of specific antibody therapy. Here we used a mathematical model to predict serotype-specific DENV prevalence and overall case burden in Mexico.
| S-EPMC10348539 | biostudies-literature