Project description:Investigation of whole genome gene expression level changes in Moyo-S and Moyo-R strains of Aedes aegypti after oral infection of serotype 1, serotype 2, serotype 3 and serotype 4 of dengue virus The Moyo-S is highly suscpetible to dengue infection whereas Moyo-R is refractory to the dengue infection. They have been investigated in our previous studies incluidng Behura et al. (2011). PLoS neglected tropical diseases 5 (11), e1385; and Chauhan et al. (2012). PloS one 7 (10), e47350.
Project description:To study the role of Dengue virus serotype 4 NS1 in modulation of host cell transcriptome we performed gene expression profiling using data obtained from RNA- sequencing of total RNA from 3 different samples: Huh7 cells (no transfection), Huh7 cells transfected with pTracer-SV40 empty vector and Huh7 cells transfected with pTracer-SV40 encoding dengue virus serotype 4 NS1 (pDENV4 NS1).
Project description:Sanofi Pasteur is developing a tetravalent YFV 17D vaccine-based chimeric dengue vaccine (TV CYD), which is currently in clinical phase 3 development. DNA microarrays were used to assess the innate gene signature in human mDCs infected with the four CYDs alone or in combination, with a wild type dengue serotype 3 virus, or with a classically attenuated serotype 3 virus (VDV3) shown to be reactogenic in humans. We observed a very reproducible signature for each of the 4 CYDs, involving stimulation of Type I IFN genes and associated genes, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, the wt DEN3 virus induced a predominantly inflammatory profile, while VDV3 appeared to induce a blunted response, which may have contributed to its clinical outcome.
Project description:Sanofi Pasteur is developing a tetravalent YFV 17D vaccine-based chimeric dengue vaccine (TV CYD), which is currently in clinical phase 3 development. DNA microarrays were used to assess the innate gene signature in human mDCs infected with the four CYDs alone or in combination, with a wild type dengue serotype 3 virus, or with a classically attenuated serotype 3 virus (VDV3) shown to be reactogenic in humans. We observed a very reproducible signature for each of the 4 CYDs, involving stimulation of Type I IFN genes and associated genes, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, the wt DEN3 virus induced a predominantly inflammatory profile, while VDV3 appeared to induce a blunted response, which may have contributed to its clinical outcome. All oligochip experiments were performed in dye swap. For each experiment, the gene expression profile of infected cells was compared with the corresponding profile of uninfected cells. 2 biologicals replicates were performed for each virus.
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.
Project description:Human primary monocytes were differentiated for 6days in the presence of M-CSF. The resulting macrophages were infected with dengue virus serotype 2, strain 16681 under the following conditions: Mock, Multiplicity of infection (MOI) 1 without antibodies, MOI 1 with 40ng/mL of irrelevant human antibody, MOI 1 with infection enhancing antibody (40ng/mL of a human monoclonal antibody against dengue), and either MOI 5 or MOI 2.5. Total cell RNA was isolated at 2h post infection or 24h post infection.
Project description:This study aims to understand the characteristics and diversification of Dengue specific immunoglobulin repertoires after different immunization strategies in mouse. We propose two different immunization strategies in dengue vaccine development. One is tetravalent strategy, which pre-mixed four serotype dengue DNA vaccine and administrated four times. The other one is sequential strategy, which sequentially administrated four time, each serotype vaccine for each dose as sequences of Den1, Den2, Den3 and Den4.
Project description:Dengue is a pan-tropic public health problem. In children, dengue shock syndrome (DSS) is the most common life-threatening complication. Predicting patients who may develop DSS may improve triage and treatment. To this end, we conducted a nested case-control comparison of the early host-transcriptional features in 24 DSS patients and 56 sex-, age-, virus serotype-matched uncomplicated dengue patients. In the first instance we defined the âearly dengueâ profile. The transcriptional signature in acute versus convalescent samples (â¤72hrs post illness-onset) was defined by an over-abundance of interferon-inducible transcripts (31% of the 551 over-abundant transcripts), and canonical gene ontology terms that included response to virus, immune response, innate immune response and inflammatory response. Pathway and network analysis identified STAT1, STAT2, STAT3, IRF7, IRF9, IRF1, CEBPB and SP1 as key transcriptional factors mediating the early response. Strikingly, the only differences in the transcriptional signatures of early DSS and uncomplicated dengue cases was the greater abundance of several neutrophil-associated transcripts in patients who progressed to DSS, a finding supported by higher plasma concentrations of several canonical proteins associated with neutrophil-degranulation (BPI, ELA2, DEF1A). Elevated levels of neutrophil-associated transcripts were independent of the neutrophil count and also the genotype of the infecting virus, as genome-length sequences of DENV-1 (n=15) and DENV-2 (n=3) viruses sampled from DSS patients were phylogenetically indistinguishable from those sampled from uncomplicated dengue patients (32 DENV-1 and 9 DENV-2). Collectively, these data suggest an hitherto unrecognised association between neutrophil activation, pathogenesis and the development of DSS and point to future strategies for guiding prognosis. 112 patients: 80 with uncomplicated dengue, 32 with dengue shock syndrome