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Resultados 1 - 20 de 22.001

Raman spectroscopy based differentiation of typhoid and dengue fever in infected human sera.

Spectrochim Acta A Mol Biomol Spectrosc; 206: 197-201, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30114579


This study investigated the potential of Raman spectroscopy, to differentiate between two different types of human pathological conditions with some symptom similarity; typhoid and dengue. Raman spectra were recorded from 20 Salmonella Typhi infected and 22 dengue virus (DENV) infected sera samples using laser wavelength 785 nm. When Raman spectrum (540 to 1830 cm ) of serum samples was compared it showed significant variations. Twelve characteristic Raman bands at 562, 649, 716, 780, 838, 1099, 1144, 1156, 1260, 1386, 1556 and 1746 cm were recorded specifically in case of the typhoid infected samples, whereas four Raman bands at 756, 1218, 1672 and 1686 cm were found specifically in the DENV infected samples. For statistical differentiation principal component analysis (PCA) and linear discriminant analysis (LDA) was applied which successfully separated two sets of data. These disease specific Raman signatures may be labeled as biomarkers for identification as well as differential diagnosis of typhoid and dengue infection. Hence we propose that Raman spectroscopy has the potential to not only identify but also to differentiate between different pathological conditions, having some similar symptoms like high grade fever in this case.

An ensemble forecast model of dengue in Guangzhou, China using climate and social media surveillance data.

Sci Total Environ; 647: 752-762, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30092532


BACKGROUND: China experienced an unprecedented outbreak of dengue in 2014, and the number of dengue cases reached the highest level over the past 25 years. There is a significant delay in the release of official case count data, and our ability to timely track the timing and magnitude of local outbreaks of dengue remains limited. MATERIAL AND METHODS: We developed an ensemble penalized regression algorithm (EPRA) for initializing near-real time forecasts of the dengue epidemic trajectory by integrating different penalties (LASSO, Ridge, Elastic Net, SCAD and MCP) with the techniques of iteratively sampling and model averaging. Multiple streams of near-real time data including dengue-related Baidu searches, Sina Weibo posts, and climatic conditions with historical dengue incidence were used. We compared the predictive power of the EPRA with the alternates, penalized regression models using single penalties, to retrospectively forecast weekly dengue incidence and detect outbreak occurrence defined using different cutoffs, during the periods of 2011-2016 in Guangzhou, south China. RESULTS: The EPRA showed the best or at least comparable performance for 1-, 2-week ahead out-of-sample and leave-one-out cross validation forecasts. The findings indicate that skillful near-real time forecasts of dengue and confidence in those predictions can be made. For detecting dengue outbreaks, the EPRA predicted periods of high incidence of dengue more accurately than the alternates. CONCLUSION: This study developed a statistically rigorous approach for near-real time forecast of dengue in China. The EPRA provides skillful forecasts and can be used as timely and complementary ways to assess dengue dynamics, which will help to design interventions to mitigate dengue transmission.

Correlation of dengue incidence and rainfall occurrence using wavelet transform for João Pessoa city.

Sci Total Environ; 647: 794-805, 2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30096669


Dengue, a reemerging disease, is one of the most important viral diseases transmitted by mosquitoes. In this study, 55,680 cases of dengue between 2007 and 2015 were reported in Paraíba State, among which, 30% were reported in João Pessoa city, with peaks in 2015, 2011 and 2013. Weather is considered to be a key factor in the temporal and spatial distribution of vector-transmitted diseases. Thus, the relationship between rainfall occurrence and dengue incidences reported from 2007 to 2015 in João Pessoa city, Paraíba State, Brazil, was analyzed by means of wavelet transform, when a frequency analysis of both rainfall and dengue incidence signals was performed. To determine the relationship between rainfall and the incidence of dengue cases, a sample cross correlation function was performed to identify lags in the rainfall and temperature variables that might be useful predictors of dengue incidence. The total rainfall within 90 days presented the most significant association with the number of dengue cases, whereas temperature was not found to be a useful predictor. The correlation between rainfall and the occurrence of dengue cases showed that the number of cases increased in the first few months after the rainy season. Wavelet analysis showed that in addition to the annual frequency presented in both time series, the dengue time series also presented the 3-year frequency from 2010. Cross wavelet analysis revealed that such an annual frequency of both time series was in phase; however, after 2010, it was also possible to observe 45° up phase arrows, which indicated that rainfall in the present year led to an increased dengue incidence the following year. Thus, this approach to analyze surveillance data might be useful for developing public health policies for dengue prevention and control.

In situ removal of consensus dengue virus envelope protein domain III fused to hydrophobin in Pichia pastoris cultures.

Protein Expr Purif; 153: 131-137, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30240632


This work describes a novel strategy for the integrated expression and purification of recombinant proteins in Pichia pastoris cultures. Hydrophobins can be used as fusion tags, proteins fused to them alter their hydrophobicity and can be purified by aqueous two-phase systems (ATPS) based on non-ionic surfactants. Here, the consensus dengue virus envelope protein domain III fused to hydrophobin I of Trichoderma reesei was expressed in Pichia pastoris cultures and an in situ product removal by an ATPS using a non-ionic detergent, (Triton X-114) was performed. The protein was produced and purified directly from the yeast culture supernatant both efficiently and with no loss. The purified protein was properly immobilized by adsorption in solid phase and recognized by anti-dengue antibodies, showing its potential for the development of an indirect immunoassay for dengue virus.

Seroprevalence of West Nile and dengue virus in the human population of the Bolivian Chaco.

J Med Virol; 91(1): 146-150, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30118540


To determine the seroprevalence of antibodies against dengue virus (DENV) and West Nile virus (WNV) in the human population of the Bolivian Chaco, we tested 256 inhabitants of two rural communities. The seroprevalence, confirmed by plaque reduction neutralization test, was 7.8% and 2.7% for DENV and WNV, respectively.

Seroprevalence of dengue fever and the associated sociodemographic, clinical, and environmental factors in Makkah, Madinah, Jeddah, and Jizan, Kingdom of Saudi Arabia.

Acta Trop; 189: 54-64, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30244133


This study aimed to estimate the seroprevalence of anti-dengue IgG antibodies in Makkah, Al Madinah, Jeddah, and Jizan; and to identify the associated demographic, clinical, and environmental independent risk factors. A community-based household serosurvey conducted between September 20, 2016 and January 31, 2017. A multi-stage stratified cluster sampling was used to select 6596 participants from Makkah, Madinah, Jeddah, and Jizan. Blood samples were drawn from all participants to detect anti-dengue IgG antibodies. A semi-structured questionnaire was used to collect information on demographic, clinical, and environmental data. Multivariate logistic regression was carried out to identify independent risk factors of dengue seropositivity. The dengue seroprevalence (95% confidence intervalI) was 26.7% (25.6%, 27.8%), with the highest (33.6%) and lowest (14.8%) rates in Jizan and Madinah, respectively, and reaching 50% or more in several districts of the four cities. Demographic predictors of seroprevalence included: dwelling in Makkah (odds ratio [OR] = 2.19, p < 0.001) or Jizan (OR = 2.17, p < 0.001); older age (OR = 3.91, p < 0.001 for age>30 years); housing type (OR = 1.84 and 1.82, p < 0.001 for popular and social houses, respectively); and number of household occupants (OR = 0.86 and 0.71 for 6-10 [p = 0.042] and 11-20 [p = 0.002] occupants, respectively). Environmental predictors included the absence of pest control works in residency area (OR = 1.39, p = 0.002), presence of mosquitoes in the home (OR = 1.39, p = 0.001), and absence of awareness campaigns (OR = 1.97, p < 0.001). One in four inhabitants of the Western region of Saudi Arabia was seropositive for the dengue virus. Implementation of behavior-based educational programs is recommended, involving the population in the identification and eradication of vector sources and promoting appropriate behaviors that prevent the spread.

Implications of meteorological and physiographical parameters on dengue fever occurrences in Delhi.

Sci Total Environ; 650(Pt 2): 2267-2283, 2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30292120


Dengue Fever has become an increasing public health concern around the world due to its serious health consequences including death, lack of effective vaccine and specific treatment. Influence of area specific meteorological and physiographical characteristics on the spread of infectious diseases need to be studied to understand spatial-temporal aspects of infectious diseases in a particular area. Mathematical relationships between various explanatory variables (causative factors) and Dengue Fever incidences have been established to quantify and prioritize the influence of various factors. So that, effective health care services could be provided in these areas. The study successfully explains the occurrences of Dengue Fever in Delhi in term of geo-spatial phenomena/variables. Meteorological data of 13 stations in Delhi at hourly temporal scale for a period 2006-2015 have been used along with multi-spectral satellite data. Data on reported cases of Dengue Fever on daily basis and for a period of ten years from 2006 to 2015 have been obtained for Delhi. Python modules have been developed to extract values of geospatial parameters and to perform Poisson regression. To assess the accuracy of developed Poisson regression based equations, r-squared and error statistics have been calculated. Results indicate strong association of Dengue Fever incidences with temperature, humidity, wind speed, sunshine hours, built-up and vegetation density and distance from dairy locations, waterbodies and drainage network. Further, critical ranges of various parameters favouring high number of Dengue Fever incidences have been determined. These findings have significant public health implications for control and prevention of Dengue Fever incidences in Delhi city and surrounding region. Occurrences of Dengue Fever incidences are found to be highest in the month of September and October. These months represent transition period from rainy season to winter season. It is recommended that further study should focus on detailed analysis of causative factors in this period.

Dengue virus potentially promotes migratory responses on endothelial cells by enhancing pro-migratory soluble factors and miRNAs.

Virus Res; 259: 68-76, 2019 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-30367889


The most life-threatening effect of the Dengue virus (DENV) infection is an acute destabilization of the microvascular endothelial cell (MEC) barrier leading to plasma leakage, hypovolemic shock and haemorrhage. However, the underlying cellular mechanisms responsible for the dysfunction of MECs are not well understood. To identify potential cellular processes altered during DENV infection of MECs, expression profiles of cytokines/growth factors and microRNAs were measured by Luminex assay and next generation sequencing, respectively. Synchronously DENV2-infected MECs increase the secretion of IL-6, IL-8, FGF-2, GM-CSF, G-CSF, TGF-α, GRO, RANTES, MCP-1 and MCP-3. Conditioned media of infected MECs increased the migration of non-infected MECs. Furthermore, six miRNAs deregulated at 24 hpi were predicted to regulate host genes involved in cell migration and vascular developmental processes such as angiogenesis. These in silico analyses provide insights that support that DENV promotes an acute migratory phenotype in MECs that contributes to the vascular destabilization observed in severe dengue cases.

Going Viral 2019: Zika, Chikungunya, and Dengue.

Dermatol Clin; 37(1): 95-105, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30466692


Chikungunya and Zika virus infections are emerging diseases in the Americas, and dengue continues to be the most prevalent arthropod-borne virus in the world. These arbovirus diseases may spread by endemic transmission or as travel-related infections and have rapidly expanded their geographic distribution secondary to vector spread. All 3 share a similar clinical picture that includes a maculopapular rash. Zika is characterized by pruritic rash, low-grade fever, and arthralgia. Congenital nervous system malformations are a growing public-health concern. Chikungunya distinctive dermatologic manifestations include facial melanosis and bullous eruption. Dengue bleeding complications may be life-threatening and require inpatient management.

Administration of plasmacytoid dendritic cell-stimulative lactic acid bacteria is effective against dengue virus infection in mice.

Int J Mol Med; 43(1): 426-434, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30365042


Dengue virus (DENV), a mosquito­borne flavivirus, causes an acute febrile illness that is a major public health problem in the tropics and subtropics globally. However, methods to prevent or treat DENV infection have not been well established. It was previously demonstrated that Lactococcus lactis strain plasma (LC­plasma) has the ability to stimulate plasmacytoid dendritic cells (pDCs). As pDCs are key immune cells that control viral infection by producing large amounts of type I interferons (IFN), the present study evaluated the effect of LC­plasma on DENV infection using a mouse infectious DENV strain. Mice were divided into two groups and the test group was orally administered LC­plasma for two weeks. Two weeks following administration, the mice were infected with DENV and the relative viral titers and the expression of the inflammatory genes in DENV­infected tissue were measured using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The relative viral titers were notably lower in the DENV­infected tissues compared with the control group when LC­plasma was orally administered prior to DENV infection. Furthermore, the expression of the inflammatory genes associated with DENV infection was also reduced by LC­plasma administration. To investigate how LC­plasma administration controls DENV infection, the present study examined anti­viral gene expression, which is critical for the viral clearance induced by type I IFN. Two weeks subsequent to the administration of LC­plasma, the expression of anti­viral gene was measured using RT­qPCR. Oral intake of LC­plasma enhanced anti­viral gene expression in DENV­infected spleen tissue. To clarify the detailed mechanism, in vitro co­culture studies using bone­marrow derived DC (BMDC) were performed. BMDC were stimulated with LC­plasma in combination with anti­IFN­α/ß antibody and the expression of anti­viral genes was measured. In vitro studies revealed that the effect of LC­plasma on anti­viral genes was dependent on type I IFN. Based on these results, LC­plasma may be effective against DENV infection by stimulating pDCs, which results in the increased production of anti­viral factors.

Effects of human mobility, temperature and mosquito control on the spatiotemporal transmission of dengue.

Sci Total Environ; 651(Pt 1): 969-978, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30360290


Dengue transmission exhibits evident geographic variations and seasonal differences. Such heterogeneity is caused by various impact factors, in which temperature and host/vector behaviors could drive its spatiotemporal transmission, but mosquito control could stop its progression. These factors together contribute to the observed distributions of dengue incidence from surveillance systems. To effectively and efficiently monitor and response to dengue outbreak, it would be necessary to systematically model these factors and their impacts on dengue transmission. This paper introduces a new modeling framework with consideration of multi-scale factors and surveillance data to clarify the hidden dynamics accounting for dengue spatiotemporal transmission. The model is based on compartmental system which takes into account the biting-based interactions among humans, viruses and mosquitoes, as well as the essential impacts of human mobility, temperature and mosquito control. This framework was validated with real epidemic data by applying retrospectively to the 2014 dengue epidemic in the Pearl River Delta (PRD) in southern China. The results indicated that suitable condition of temperature could be responsible for the explosive dengue outbreak in the PRD, and human mobility could be the causal factor leading to its spatial transmission across different cities. It was further found that mosquito intervention has significantly reduced dengue incidence, where a total of 52,770 (95% confidence interval [CI]: 29,231-76,308) dengue cases were prevented in the PRD in 2014. The findings can offer new insights for improving the predictability and risk assessment of dengue epidemics. The model also can be readily extended to investigate the transmission dynamics of other mosquito-borne diseases.

Dengue Virus IgM Serotyping by ELISA with Recombinant Mutant Envelope Proteins.

Emerg Infect Dis; 25(1)2019 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-30387417


We developed an IgM-based ELISA that identifies the dengue virus serotype of recent infections. Dominant serotypes were detectable in 91.1% of samples from travelers and 86.5% of samples from residents of endemic regions; 97.1% corresponded to the serotype identified by PCR. This ELISA enables more accurate reporting of epidemiologic findings.

Optimization of a fragment linking hit toward Dengue and Zika virus NS5 methyltransferases inhibitors.

Eur J Med Chem; 161: 323-333, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30368131


No antiviral drugs to treat or prevent life-threatening flavivirus infections such as those caused by mosquito-borne Dengue (DENV) and more recently Zika (ZIKV) viruses are yet available. We aim to develop, through a structure-based drug design approach, novel inhibitors targeting the NS5 AdoMet-dependent mRNA methyltransferase (MTase), a viral protein involved in the RNA capping process essential for flaviviruses replication. Herein, we describe the optimization of a hit (5) identified using fragment-based and structure-guided linking techniques, which binds to a proximal site of the AdoMet binding pocket. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 30 and 33 (DENV IC = 26 µM and 23 µM; ZIKV IC = 28 µM and 19  µM, respectively), two representatives of novel non-nucleoside inhibitors of flavivirus MTases.

The spread of mosquito-borne viruses in modern times: A spatio-temporal analysis of dengue and chikungunya.

Spat Spatiotemporal Epidemiol; 26: 113-125, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30390927


Since the 1970s, mosquito-borne pathogens have spread to previously disease-free areas, as well as causing increased illness in endemic areas. In particular, dengue and chikungunya viruses, transmitted primarily by Aedes aegypti and secondarily by Aedes albopictus mosquitoes, represent a threat for up to a third of the world population, and are a growing public health concern. In this study, we assess the spatial and temporal factors related to the occurrences of historic dengue and chikungunya outbreaks in 76 nations focused geographically on the Indian Ocean, with outbreak data from 1959 to 2009. First, we describe the historical spatial and temporal patterns of outbreaks of dengue and chikungunya in the focal nations. Second, we use a boosted regression tree approach to assess the statistical relationships of nations' concurrent outbreak occurrences and annual occurrences with their spatial proximity to prior infections and climatic and socio-economic characteristics. We demonstrate that higher population density and shorter distances among nations with outbreaks are the dominant factors that characterize both dengue and chikungunya outbreaks. In conclusion, our analysis provides crucial insights, which can be applied to improve nations' surveillance and preparedness for future vector-borne disease epidemics.

Caso fatal de coinfectada de rickettsiosis y virus del dengue en México / Fatal case of co-infected of rickettiosis and dengue virus in Mexico

Rev Med Inst Mex Seguro Soc; 56(3): 320-322, 2018 Oct 25.
Artigo em Espanhol | MEDLINE | ID: mdl-30394722


Background: Rocky Mountain spotted fever (RMSF) is a disease spread by an infected tick and it is lethal if patient is not treated on time. Symptom similarities with other exanthematous diseases may delay the diagnosis, which leads to its mortality. Clinical case: We show the lethal case of a patient with medical record of high blood pressure and no history of travel, who lived in Sonora, Mexico. At the beginning, it was suspected that the patient had a dengue virus infection, which was confirmed positive by an ELISA test. Patient's rapid deterioration, multi-organic failure and the characteristics of her exanthema led to the suspicion of the presence of RMSF. We started doxycycline treatment; however, patient died after seven days of evolution. It was confirmed a co-infection of Rickettssia spp. and dengue virus. Conclusion: In rickettsial endemic zones, treatment with doxycycline should be employed in patients with similar symptoms, even though there is evidence of the presence of other etiologic agents.

Dengue Virus Infection of Primary Human Smooth Muscle Cells.

Am J Trop Med Hyg; 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30398136


Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMCs) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVECs) and the classically used, non-transformed, and highly permissive LLC-MK2 cells (macaque kidney cells). Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection.

Factors Associated with Dengue Virus Infection and Reinfection in Asymptomatic Children in Two Colombian Municipalities.

Am J Trop Med Hyg; 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30398143


Dengue is the most important arbovirosis in the world. In this study, we assessed the knowledge, attitudes, and practices (KAP) regarding dengue in parents from two small Colombian municipalities in the Cundinamarca Province. Parents and their healthy children from 4 to 14 years of age were included in some public elementary schools. After a medical examination, blood samples were taken for diagnosis of dengue using enzyme-linked immunosorbent assays (capture IgM and IgG, and indirect IgG and NS1) and detection of viral RNA by reverse transcription polymerase chain reaction. In addition, a KAP survey was applied to the children's parents or tutors. The indirect IgG test determined that of the 347 examined children, 87.9% had a previous infection with the dengue virus (DENV), 12.7% of them were positive for viral RNA (asymptomatic infection), and 32.0% presented reinfections. Risk factors evaluation showed that children aged 8 years and older living in the municipalities for more than 7 years were more likely to be infected or reinfected by DENV. In the same way, poor nutrition, lack of water supply, sewer service, or waste disposal services could raise the likelihood of dengue infections. The surveys indicated that parents have unhealthy practices and a low knowledge about the transmission of the disease, which could result in an increase of mosquito breeding sites, allowing sustained dengue transmission.

Ezetimibe inhibits dengue virus infection in Huh-7 cells by blocking the cholesterol transporter Niemann-Pick C1-like 1 receptor.

Antiviral Res; 160: 151-164, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30391500


Despite the importance of Dengue virus (DENV) infection in human health, there is not a fully effective vaccine or antiviral treatment against the infection. Since lipids such as cholesterol are required during DENV infection, its uptake and synthesis are increased in infected cells. Ezetimibe is an FDA-approved drug that reduces cholesterol uptake by inhibiting the endocytosis through Niemman-Pick C1-Like 1 (NPC1L1) receptor, expressed on the membrane of enterocytes and hepatocytes. Our results indicate that an increase in the amount of NPC1L1 occurs on the surface of Huh-7 cells during DENV infection, which correlates with an increase in cholesterol levels. Blockage of NPC1L1 with ezetimibe in concentrations up to 50 µM does not reduce cell viability but diminished total cellular cholesterol, the percentage of infected cells, viral yield, viral RNA and protein synthesis without affecting DENV binding and/or entry to Huh-7 cells. Moreover, ezetimibe inhibited DENV replicative complex formation and lipid droplets accumulation. All these results indicate that ezetimibe is an excellent drug to inhibit DENV infection and confirm that cholesterol is a key target to inhibit viral infection.

Induction of anti-viral genes mediated by humoral factors upon stimulation with Lactococcus lactis strain plasma results in repression of dengue virus replication in vitro.

Antiviral Res; 160: 101-108, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30393011


Dengue is a mosquito-borne disease caused by dengue virus (DENV) infection. There is currently no effective vaccine or antiviral treatment available against DENV. In previous studies, we showed that Lactococcus lactis strain Plasma (LC-Plasma) could activate plasmacytoid dendritic cells, which play an important role against virus infection. LC-Plasma administration ameliorated symptoms of viral diseases and its effect appeared to be associated with IFN-α induction. However the precise mechanism of LC-Plasma protection remained unclear. In this study, we investigated the effects of LC-Plasma-induced humoral factors on DENV replication using HepG2 cells as an in vitro infection model. When HepG2 cells were preincubated with supernatants of LC-Plasma-stimulated bone marrow-derived dendritic cells, the replication of DENV was significantly inhibited in a dose dependent manner and its activity was evident regardless of the DENV serotype. In addition, the expression of interferon-stimulated genes, including ISG15, IFITM-1, MxA, RSAD2, and RyDEN, was significantly upregulated by humoral factors. We also compared the effects of representative strains of lactic acid bacteria and found that the ability to prevent DENV replication was unique to LC-Plasma. In addition, it was revealed that both anti-DENV replication activity and ISG induction depended on type I IFN rather than type III IFN signaling. Taken together, since LC-Plasma induces, in a more natural form, potent anti-DENV replication activities irrespective of viral serotypes via induction of type I IFN, LC-Plasma could be safely used as a prophylactic anti-DENV option.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction.

J Vis Exp; (141)2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30451226


At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.
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