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Rev. Ciênc. Méd. Biol. (Impr.) ; 21(1): 40-45, maio 05,2022. fig
Article in Portuguese | LILACS | ID: biblio-1370563


Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Animals , Mice , Dengue/immunology , Dengue Virus/immunology , Antibodies, Monoclonal/biosynthesis , Antigens, Viral/administration & dosage , Injections, Intraperitoneal , Mice, Inbred BALB C
Braz. j. infect. dis ; 24(1): 85-88, Feb. 2020. tab, graf
Article in English | LILACS | ID: biblio-1089325


ABSTRACT The antigenic potential of seven immunogenic peptides of the dengue virus was evaluated in the sera of patients with dengue confirmed by IgM/IgG serology. Antibodies IgM and IgG against dengue virus peptides were analyzed by ELISA in 31 dengue sero-positive and 20 sero-negative patients. The P5 peptide showed significant IgG immunoreactivity mostly in the sera of patients with dengue without warning signs in comparison with patients with dengue with warning signs, correlating with mild disease. This finding suggests that the low antibody response against P5 epitope could be a risk factor for higher susceptibility to dengue virus infection with warning signs, and that P5 could be a potential antigen for vaccine development.

Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Aged , Young Adult , Peptides/immunology , Viral Envelope Proteins/immunology , Dengue Virus/immunology , Dengue Vaccines , Antibodies, Viral/immunology , Epitopes/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Enzyme-Linked Immunosorbent Assay , Statistics, Nonparametric , Dengue/immunology , Dengue/prevention & control , Antibody Formation , Antigens, Viral/immunology
Chinese Journal of Biotechnology ; (12): 2206-2215, 2020.
Article in Chinese | WPRIM | ID: wpr-878479


Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.

Animals , Humans , Mice , Antibodies, Monoclonal , Antibodies, Viral/metabolism , Dengue/diagnosis , Dengue Virus/immunology , Enzyme-Linked Immunosorbent Assay , Mice, Inbred BALB C , Sensitivity and Specificity , Serogroup , Viral Nonstructural Proteins/immunology
Mem. Inst. Oswaldo Cruz ; 115: e200225, 2020.
Article in English | LILACS, SES-SP | ID: biblio-1135239


In the near future, the overlap of Coronavirus disease 2019 (COVID-19) and dengue epidemics is a concrete threat in tropical regions. Co-epidemics of COVID-19 and dengue could be an overwhelming challenge for health systems in low- and middle-income countries. In this work, we investigated potential serological cross-reactions between COVID-19 and dengue patients. Among 32 COVID-19 positive sera, no positive Dengue virus (DENV) IgG/IgM results were observed. On the other hand, one false-positive result was observed among 44 DENV-positive sera tested for COVID-19 antibodies with each of the two rapid tests used. Further data on accuracy of COVID-19 diagnostic test are urgently warranted.

Humans , Pneumonia, Viral/immunology , Coronavirus Infections/immunology , Cross Reactions , Dengue/immunology , Antibodies, Viral/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Enzyme-Linked Immunosorbent Assay , Dengue Virus/immunology , Pandemics , Betacoronavirus/immunology , SARS-CoV-2 , COVID-19
Mem. Inst. Oswaldo Cruz ; 115: e200287, 2020. tab, graf
Article in English | LILACS | ID: biblio-1154869


BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

Humans , Immunologic Tests/methods , Enzyme-Linked Immunosorbent Assay/methods , Viral Nonstructural Proteins/immunology , Reverse Transcriptase Polymerase Chain Reaction/methods , Dengue/diagnosis , Dengue Virus/isolation & purification , Antigens, Viral/blood , Predictive Value of Tests , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics , Dengue/blood , Dengue/virology , Dengue Virus/genetics , Dengue Virus/immunology , Antibodies, Viral/blood , Antigens, Viral/immunology
Rev. cuba. med. trop ; 71(1): e338, ene.-abr. 2019. tab
Article in Spanish | LILACS, CUMED | ID: biblio-1093549


Los arbovirus constituyen una de las principales causas de emergencia en salud por la morbilidad y mortalidad que producen y el estrés sanitario que conllevan. Cuba no ha estado excenta de riesgo, y el enfrentamiento del dengue inicialmente y de otros arbovirus después, ha sido, y es, una prioridad de las máximas autoridades del país. La vigilancia de laboratorio de dengue se estableció desde inicios de la década del 70 aunque sus objetivos y estrategias han cambiado según la situación epidemiológica nacional y regional y la tecnología de diagnóstico disponible. Se destacan cuatro etapas en su desarrollo. En este trabajo se resumen las estrategias desarrolladas para la vigilancia de laboratorio de dengue y de otros arbovirus en el periodo de 1970 a 2017. Se describe además el papel desempeñado por el Instituto de Medicina Tropical, ¨Pedro Kouri¨ (IPK) como Laboratorio Nacional de Referencia(AU)

Arboviruses are one of the leading causes of health emergencies due to their morbidity and mortality and the sanitary stress they bring about. Cuba has not been free from risk, and the response first to dengue fever and then to other arboviruses has been and still is a priority for the country's top authorities. Laboratory surveillance of dengue fever was implemented in the 1970s, though its aims and strategies have evolved in keeping with the national and regional epidemiological situation, and the available diagnostic technology. Four stages stand out in the development of dengue laboratory surveillance. The present paper summarizes the strategies developed for laboratory surveillance of dengue fever and other arboviruses in the period 1970-2017. A description is also provided of the role played by Pedro Kourí Tropical Medicine Institute (IPK) as a national reference laboratory(AU)

Humans , Arbovirus Infections/prevention & control , Surveillance in Disasters , Dengue/epidemiology , Dengue Virus/immunology , Public Health Laboratory Services
Rev. Soc. Bras. Med. Trop ; 51(4): 503-507, July-Aug. 2018. tab, graf
Article in English | LILACS | ID: biblio-1041474


Abstract INTRODUCTION: Socioeconomic disparities in the community make some groups more vulnerable to dengue infection. METHODS: Fourteen dengue cases (IgM positive) served as index cases for the positive geographic cluster investigations. RESULTS: Of 292 individuals, the overall dengue seroprevalence was 22.9% (IgM positive 4.8%; IgG positive 18.1%). The highest (45%) seroprevalence was reported in the most socioeconomically vulnerable lower class, followed by the middle class (39%). Orthogonal comparisons showed that socioeconomic factors play a significant role in the prevalence of dengue. CONCLUSIONS: An integrated approach is required to control the menace through vector control strategies and improvement of socioeconomic conditions.

Humans , Male , Female , Adolescent , Adult , Young Adult , Dengue/epidemiology , Dengue Virus/immunology , Antibodies, Viral/blood , Pakistan/epidemiology , Socioeconomic Factors , Immunoglobulin G/blood , Immunoglobulin M/blood , Seroepidemiologic Studies , Prevalence , Cross-Sectional Studies , Middle Aged
Rev. Soc. Bras. Med. Trop ; 51(2): 168-173, Mar.-Apr. 2018. graf
Article in English | LILACS | ID: biblio-897058


Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Humans , Leukocytes, Mononuclear/virology , Dengue/diagnosis , Dengue Virus/genetics , Dengue Virus/immunology , Antibodies, Viral/blood , Cell Separation , Chromatography, Affinity , Sensitivity and Specificity , Viral Nonstructural Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Flow Cytometry , Fluorescence
Medicina (B.Aires) ; 78(1): 23-28, feb. 2018.
Article in Spanish | LILACS | ID: biblio-894542


Dengue (DENV), zika (ZIKV) y chikungunya (CHIKV), tres arbovirosis transmitidas por mosquitos Aedes, se han propagado en las últimas décadas en zonas tropicales y subtropicales húmedas. El dengue es epidémico en áreas subtropicales de la Argentina. Después de la infección por DENV hay inmunidad duradera contra el serotipo infectante, pero aumenta el riesgo de enfermedad grave por los otros tres. La vacuna recombinante tetravalente, Dengvaxia® previene el dengue grave y la hospitalización en sujetos seropositivos. En 2017 se aprobó Dengvaxia en Argentina, para edades de 9 a 45 años, sin incluirla en el calendario nacional de vacunación. Otras dos vacunas se hallan en evaluación Fase III: la desarrollada por NIAID/ Instituto Butantan y la vacuna Takeda. ZIKV, virus asociado a microcefalia en recién nacidos en Brasil, circula desde 2016 en Argentina. Aún no existe vacuna de actividad comprobada contra ZIKV ni tratamiento eficaz. No se registró circulación activa de CHIKV en Argentina en 2017. Los brotes de fiebre CHIKV tienen una complicación: el desarrollo de reumatismo crónico post-enfermedad. No existen vacunas aprobadas para humanos ni terapias antivirales efectivas. La gravedad de estas virosis contribuyó a un rápido progreso en el conocimiento de los procesos de infección y de la respuesta inmune. Pero sus vectores, Aedes aegypti y A. albopictus, continúan expandiéndose, lo que indica que la vacuna será el medio más efectivo para el control. Se resume aquí información sobre estas arbovirosis en Argentina y Brasil, y se describen avances en el desarrollo y la evaluación de vacunas.

Dengue (DENV), zika (ZIKV) and chikungunya (CHIKV), three arbovirosis transmitted by Aedes mosquitoes, have spread in recent decades in humid tropical and subtropical zones. Dengue is epidemic in subtropical areas of Argentina. DENV infection confers lasting immunity against the infecting serotype but increases the risk of serious disease upon reinfection by any of the other three. The recombinant tetravalent vaccine Dengvaxia® prevents severe dengue and hospitalization in seropositive subjects. In 2017, Dengvaxia was approved in Argentina, for ages 9 to 45, but is not included in the national vaccination calendar. Two other vaccines are in Phase III evaluation: one developed by NIAID / Instituto Butantan and the other by Takeda.ZIKV, a virus associated with microcephaly in newborns in Brazil, circulates since 2016 in Argentina. There is still not effective treatment nor vaccine with proven activity against ZIKV. There has been no active circulation of CHIKV in Argentina in 2017. Outbreaks of CHIKV fever have a complication: the development of chronic post-disease rheumatism. There are not approved vaccines for humans nor effective antiviral therapies. The seriousness of these virosis has contributed to a rapid progress in the knowledge of the infection processes and the immune response. For now, Aedes aegypti and A. albopictus vectors continue to expand, suggesting that the vaccine will be the most effective means of controlling these viruses. Here we summarize information about these arbovirosis in Argentina and Brazil and describe advances in the development and evaluation of vaccines.

Humans , Child , Adolescent , Adult , Young Adult , Dengue/prevention & control , Chikungunya Fever/prevention & control , Zika Virus Infection/prevention & control , Argentina/epidemiology , Brazil/epidemiology , Viral Vaccines/administration & dosage , Chikungunya virus/immunology , Dengue/epidemiology , Dengue Virus/immunology , Dengue Vaccines/administration & dosage , Chikungunya Fever/epidemiology , Zika Virus/immunology , Zika Virus Infection/epidemiology
Mem. Inst. Oswaldo Cruz ; 113(11): e170538, 2018. tab
Article in English | LILACS | ID: biblio-1040584


This study showed that laboratory markers of recent infection by dengue, Zika or chikungunya arboviruses were detected in the biological samples of approximately one-third of patients with encephalitis, myelitis, encephalomyelitis or Guillain-Barré syndrome, in a surveillance programme in Piauí state, Brazil, between 2015-2016. Fever and myalgia had been associated with these cases. Since in non-tropical countries most infections or parainfectious diseases associated with the nervous system are attributed to herpesviruses, enteroviruses, and Campylobacter jejuni, the present findings indicate that in tropical countries, arboviruses may now play a more important role and reinforce the need for their surveillance and systematic investigation in the tropics.

Humans , Chikungunya virus/genetics , Chikungunya virus/immunology , Dengue Virus/genetics , Dengue Virus/immunology , Zika Virus/genetics , Zika Virus/immunology , Acute Disease , Reverse Transcriptase Polymerase Chain Reaction , Guillain-Barre Syndrome/diagnosis , Guillain-Barre Syndrome/virology , Encephalitis/diagnosis , Encephalitis/virology , Encephalomyelitis, Acute Disseminated/diagnosis , Encephalomyelitis, Acute Disseminated/virology , Enzyme-Linked Immunospot Assay , Myelitis, Transverse/diagnosis , Myelitis, Transverse/virology , Nervous System Diseases/diagnosis , Nervous System Diseases/virology
Mem. Inst. Oswaldo Cruz ; 112(12): 829-837, Dec. 2017. tab, graf
Article in English | LILACS | ID: biblio-894854


BACKGROUND Dengue viruses (DENV) are considered one of the most important emerging pathogens and dengue disease is a global health threat. The geographic expansion of dengue viruses has led to co-circulation of all four dengue serotypes making it imperative that new DENV control strategies be devised. OBJECTIVES Here we characterize dengue serotype-specific innate immune responses in Aedes aegypti and Aedes albopictus using DENV from Puerto Rico (PR). METHODS Ae. aegypti and Ae. albopictus were infected with dengue serotype 1 and 2 isolated from Puerto Rico. DENV infected mosquito samples were collected and temporal change in expression of selected innate immune response pathway genes analyzed by quantitative real time PCR. FINDINGS The Toll pathway is involved in anti-dengue response in Ae. aegypti, and Ae. albopictus. Infections with PR DENV- 1 elicited a stronger response from genes of the Toll immune pathway than PR DENV-2 in Ae. aegypti but in infected Ae. albopictus expression of Toll pathway genes tended to be similar between the serotypes. Two genes (a ribosomal S5 protein gene and a nimrod-like gene) from Ae. albopictus were expressed in response to DENV. MAIN CONCLUSIONS These studies revealed a role for antiviral genes in DENV serotype-specific interactions with DENV vectors, demonstrated that infections with DENV-2 can modulate the Toll immune response pathway in Ae. aegypti and elucidated candidate molecules that might be used to interfere with serotype specific vector-virus interactions.

Animals , Aedes/classification , Aedes/virology , Dengue Virus/isolation & purification , Dengue Virus/genetics , Dengue Virus/immunology , Insect Vectors/classification , Insect Vectors/virology , Real-Time Polymerase Chain Reaction
Rev. Soc. Bras. Med. Trop ; 50(3): 379-382, May-June 2017. tab
Article in English | LILACS | ID: biblio-1041413


Abstract INTRODUCTION: The incidence of dengue has increased throughout the 2000s with a consequent global increase in atypical clinical forms. METHODS: This study reports a series of cases of neurological dengue out of 498 confirmed cases of laboratory dengue in Goiânia, Brazil. Cases were confirmed based on viral RNA detection via polymerase chain reaction or IgM antibody capture. RESULTS: Neurological symptoms occurred in 5.6% of cases, including paresthesia (3.8%), encephalitis (2%), encephalopathy (1%), seizure (0.8%), meningoencephalitis (0.4%), and paresis (0.4%). DENV-3 was the predominant circulating serotype (93%). CONCLUSIONS: We reported dengue cases with neurological manifestations in endemic area.

Humans , Male , Female , Aged , Paresthesia/virology , Enzyme-Linked Immunosorbent Assay , Encephalitis, Viral/virology , Dengue/complications , Dengue/epidemiology , Meningoencephalitis/virology , Paresthesia/epidemiology , Brazil/epidemiology , RNA, Viral/genetics , Polymerase Chain Reaction , Encephalitis, Viral/epidemiology , Dengue Virus/genetics , Dengue Virus/immunology , Meningoencephalitis/epidemiology , Middle Aged , Antibodies, Viral/blood
Rev. chil. infectol ; 34(3): 249-256, jun. 2017. ilus, tab
Article in Spanish | LILACS | ID: biblio-899708


Dengue fever, caused by dengue virus (DENV) infection, is one of the most important diseases in the world, not only due to the high morbidity/mortality rates it causes, but also because of its great economic and social impact in tropical/subtropical countries. DENV infection has a wide range of clinical manifestations ranging from asymptomatic infection or infection with mild symptoms to severe dengue that can lead to death. At present, no etiological treatment or effective globally distributed vaccine against the four DENV serotypes exists. Despite great efforts made to understand the mechanism associated with DENV disease pathogenesis the causes leading to severe dengue presentation have not been clarified. Some hypotheses seek to give a biological and physiological explanation to the clinical manifestations that appear during the infection. Based on the evidence that after contact with dendritic cells DENV alters the functionality of these cells, this review aims to describe the most relevant findings regarding the importance of dendritic cells in the context of DENV infection and progression of the illness.

El dengue, causada por el virus dengue (DENV), es una de las enfermedades más importantes no sólo por los altos índices de morbilidad/mortalidad, sino también por su gran impacto económico y social en los países de las regiones tropicales/subtropicales. La infección por el DENV cursa por un variado rango de manifestaciones clínicas que van desde una infección asintomática o con síntomas leves, hasta el dengue grave que puede ser fatal. En la actualidad, no se dispone de un tratamiento etiológico y tampoco de una vacuna eficaz mundialmente distribuida, contra los 4 serotipos del DENV. A pesar de los grandes esfuerzos orientados a entender el mecanismo asociado con la patogénesis de la enfermedad, aún no se ha logrado esclarecer de forma definitiva las causas que conllevan a las formas graves de enfermedad. Algunas hipótesis buscan dar una explicación biológica y fisiológica a las manifestaciones clínicas que se presentan durante la infección. Dado que una de ellas sugiere que luego del contacto con las células dendríticas el DENV altera su funcionalidad, la presente revisión tiene como objetivo describir los hallazgos más relevantes referentes a la importancia de dichas células en el marco de la infección por el DENV y progresión de la enfermedad.

Humans , Virus Replication/immunology , Dendritic Cells/immunology , Dengue/immunology , Dengue Virus/immunology , Disease Progression , Immunity, Cellular , Immunity, Innate
Mem. Inst. Oswaldo Cruz ; 112(6): 458-468, June 2017. tab, graf
Article in English | LILACS | ID: biblio-841802


ABSTRACT BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features. METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry. FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAIN CONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

Humans , Antiviral Agents/pharmacology , Plant Extracts/pharmacology , Cell Survival/drug effects , Cytokines/drug effects , Cytokines/immunology , Chemokines/drug effects , Chemokines/immunology , Uncaria/chemistry , Dengue/physiopathology , Dengue/immunology , Dengue/virology , Dengue Virus/drug effects , Dengue Virus/immunology , Antigens, Viral/drug effects , Antigens, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 15(1): 7-15, abr. 2017. ilus
Article in Spanish | LILACS, BDNPAR | ID: biblio-1008720


Los flavivirus son responsables de una considerable morbi-mortalidad a nivel mundial. Entre ellos, el virus del dengue (DENV) es causante de graves problemas de salud pública en Paraguay. El objetivo del estudio fue detectar infecciones por flavivirus a través de una reacción de RT-nested PCR genérica para flavivirus en 195 muestras de individuos con sospecha de dengue, negativos por el test inmunocromatográfico (antígeno NS1 ­ DENV), provenientes del área metropolitana de Asunción entre 2011 y 2013. Las muestras positivas para flavivirus fueron sometidas a dos reacciones de RT-nested PCRs específicas para DENV. El límite de detección (LD) para flavivirus fue de 0,2 UFP/reacción. En total 43/195 muestras fueron positivas para flavivirus. De estas, 38/43 (88,4%) correspondieron a DENV (6 DENV-1, 30 DENV-2 y 2 DENV-3). Además, 5/43 casos (11,6%) positivos para flavivirus fueron negativos para DENV por ambas reacciones específicas, pudiendo deberse a infecciones por otros flavivirus. Los resultados sugieren que la utilización de una reacción genérica seguida de otras reacciones específicas para DENV en casos febriles negativos para NS1 por el método inmunocromatográfico permitiría detectar más casos de infecciones por DENV y además, podría contribuir a la identificación de casos debido a infecciones por otros flavivirus.

Flaviviruses are responsible for considerable worldwide morbidity and mortality. Among them, the dengue virus (DENV) causes serious public health problems in Paraguay. The objective of the study was to detect flavivirus infections using a generic RT-nested -PCR in 195 samples of individuals with suspected dengue and negative for the inmunochromatographic test (NS1 antigen ­ DENV), from the metropolitan area of Asuncion between 2011 and 2013. The flavivirus-positive samples were subjected to two reactions of DENV-specific RT-nested PCRs. The detection limit (DL) for flavivirus was 0.2 PFU / reaction. In total, 43/195 samples were positive for flavivirus. Of them, 38/43 (88,4%) corresponded to DENV (6 DENV-1, 30 DENV-2 and 2 DENV-3). In addition, 5/43 cases (11.6%) positive for flavivirus were negative for DENV by both specific reactions, and may be infections caused by other flaviviruses. The results suggest that the use of a generic reaction followed by other DENV specific reactions in febrile negative cases for NS1 by the immunochromatographic method would allow the detection of more cases of DENV infections and could contribute to the identification of cases due to infections by others flaviviruses.

Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Aged, 80 and over , Flavivirus Infections/diagnosis , Dengue Virus/isolation & purification , Flavivirus/isolation & purification , Paraguay , Cross-Sectional Studies , Genome, Viral , Reverse Transcriptase Polymerase Chain Reaction , Dengue Virus/genetics , Dengue Virus/immunology , Fever , Flavivirus/genetics , Antigens, Viral/isolation & purification
Biomédica (Bogotá) ; 37(1): 34-41, ene.-feb. 2017. tab
Article in Spanish | LILACS | ID: biblio-888441


Resumen Introducción: El dengue representa un grave problema de salud pública para Colombia y, en el departamento del Quindío, afecta el 90 % de los municipios. Se necesitan estudios actualizados sobre la seroprevalencia en la población general para reforzar las acciones de salud pública. Objetivo: Determinar la seroprevalencia de la infección por dengue en barrios con alta incidencia de dengue en cuatro municipios del departamento del Quindío: Armenia, Calarcá, La Tebaida y Montenegro, en 2014. Materiales y métodos: Se hizo un estudio de prevalencia mediante muestreo probabilístico estratificado y bietápico. Se hizo una encuesta a 658 sujetos residentes del área urbana de los municipios seleccionados y se les tomó una muestra de sangre por venopunción para determinar anticuerpos IgG e IgM contra el virus del dengue. Resultados: La seroprevalencia de anticuerpos IgG en el Quindío fue de 89,4 %; en Armenia fue de 88,7 %, en Calarcá, de 81,5 %, en Montenegro, de 91,8 %, y en La Tebaida, de 97,8 %. La seroprevalencia de anticuerpos IgM en Quindío fue de 14,2 %; en Armenia, de 11,5 %, en Calarcá, de 13,0 %, en Montenegro, de 13,1%, y en La Tebaida, de 28,9 %. Conclusiones: Se evidenció una alta prevalencia de anticuerpos IgG e IgM en los cuatro municipios. En todos los grupos de edad se encontraron personas seropositivas para IgM, lo cual indicaría infección reciente. La seropositividad simultánea para IgM e IgG (12,9 %) puede indicar infección secundaria por otro serotipo del virus o una infección en los tres meses anteriores. Es necesario impulsar estrategias multisectoriales para el control de la transmisión del dengue en el Quindío.

Abstract Introduction: Dengue is a serious public health problem in Colombia; it is prevalent in 90% of the municipalities in Quindío. Studies on its seroprevalence are required to address public health interventions. Objective: To establish the seroprevalence of dengue infection in neighborhoods with high incidence in the municipalities of Armenia, Calarcá, La Tebaida and Montenegro, Quindío, in 2014. Materials and methods: We conducted a probabilistic, stratified, two-stage prevalence study. We interviewed 658 residents in the urban area of the selected municipalities. After they signed the informed consent, we took a blood sample to determine dengue IgG and IgM antibodies. Results: Seroprevalence of IgG in Quindío was 89,4%; in Armenia it was 88,7%, in Calarcá, 81,5%, in Montenegro, 91,8% and in La Tebaida 97,8%. IgM was 14, 2% in Quindío; in Armenia it was 11,5%, in Calarcá, 13,0%, in Montenegro, 13,1% and in La Tebaida, 28,9%. Conclusions: We found a high prevalence of both IgG and IgM in the four municipalities. We had positive results for IgM in all age groups, which suggests recent infection. We also found simultaneous seropositivity for IgG and IgM (12.9%), which may indicate infection by another serotype or presence of infection in the past three months. A multisectoral approach is necessary for dengue control in Quindío.

Humans , Immunoglobulin G/blood , Dengue/epidemiology , Dengue Virus/immunology , Antibodies, Viral/blood , Armenia/epidemiology , Seroepidemiologic Studies , Incidence , Prevalence , Cities , Dengue/blood , Montenegro/epidemiology
Biomédica (Bogotá) ; 37(1): 131-140, ene.-feb. 2017. graf
Article in Spanish | LILACS | ID: biblio-888451


Resumen Introducción: El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo: Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos: Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados: Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión: Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.

Abstract Introduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.

Animals , Humans , Mice , Virus Replication/physiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Dengue/virology , Dengue Virus/immunology , Antibodies, Viral/immunology , Virus Replication/genetics , Virus Replication/immunology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Endopeptidases/chemistry , Blotting, Western , Viral Nonstructural Proteins/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/chemistry , Antibodies, Viral/metabolism , Antibodies, Viral/chemistry
Rev. chil. infectol ; 33(4): 452-454, ago. 2016.
Article in Spanish | LILACS | ID: biblio-1042622


Dengue was first diagnosed on Easter Island on year 2002 and thereafter recurrent outbreaks have occurred involving different serotypes of dengue virus. Its vector, Aedes aegypti has not been eliminated despite the small size of the island. Conditions at the local hospital preclude adequate management of severe and hemorrhagic cases due to the absence of a Critical Care Unit as well as no availability of platelets, or plasma units for transfusion. Besides, transfer, of severely affected patients to continental Chile is cumbersome, slow and expensive. In this scenario, it is advisable to implement selective vaccination of Easter Island habitants with an available quadrivalent attenuated dengue vaccine with the aim to reduce hemorrhagic and severe dengue cases. This strategy should not replace permanent efforts to control waste disposal sites, water sources, maintain vector surveillance and increase education of the population.

El dengue surgió el año 2002 en Isla de Pascua y se ha presentado en brotes intercurrentes desde entonces con aparición de diferentes serotipos. El vector Aedes aegypti no ha logrado ser eliminado a pesar del pequeño tamaño de la isla y las condiciones del hospital local no permiten el manejo de casos graves por ausencia de una unidad de cuidados intensivos y disponibilidad de transfusiones de plaquetas o plasma fresco congelado. Además, el traslado de pacientes graves hacia el continente no es inmediato y es muy costoso. En este escenario, es aconsejable vacunar selectivamente a la población residente con la vacuna cuadrivalente atenuada para disminuir la probabilidad de dengue grave. Esta estrategia no debe reemplazar los esfuerzos para el control de basurales, fuentes de agua, vigilancia del vector y educación de la población.

Humans , Animals , Male , Female , Child , Adolescent , Adult , Middle Aged , Young Adult , Vaccination , Dengue/prevention & control , Dengue Vaccines/therapeutic use , Polynesia/epidemiology , Advisory Committees , Dengue/transmission , Dengue/epidemiology , Dengue Virus/immunology , Insect Vectors/virology
Medicina (B.Aires) ; 76(2): 93-97, abr. 2016.
Article in Spanish | LILACS | ID: biblio-841549


Los responsables de la actual pandemia de Chikungunya (alfavirus), dengue y Zika (flavivirus) son virus trasmitidos por artrópodos, arbovirus. Su importancia aumentó en las Américas en los últimos 20 años. Los vectores principales son Aedes aegypti y A. albopictus. La infección por dengue provee inmunidad duradera al serotipo específico y temporaria a otros tres. La posterior infección por otro serotipo determina mayor gravedad. Existe una vacuna contra dengue registrada, Dengvaxia (Sanofi Pasteur). Otras dos (Butantan y Takeda) comienzan la Fase III en 2016. La infección por Zika suele ser asintomática, o presentarse con exantema, conjuntivitis y fiebre no muy elevada. No existen vacunas ni tratamiento específico. Se puede transmitir por vía parental, sexual y por transfusión sanguínea. Se la ha asociado con microcefalia. Chikungunya causa artralgias prolongadas, con respuesta inmune persistente. Hay dos vacunas candidatas en Fase II. El diagnóstico directo del dengue se realiza por cultivo, RT-PCR y ELISA para detección del antígeno NS1; los métodos indirectos son ELISA-IgM (reacción cruzada con otros flavivirus), MAC-ELISA, y neutralización en placas, que diferencia los 4 serotipos DENV y otros flavivirus. Zika se diagnostica por RT-PCR y aislamiento del virus. El diagnóstico serológico presenta reacciones cruzadas con otros flavivirus. Para CHIKV se emplean cultivo y RT-PCR, MAC-ELISA y neutralización en placas. Contra Aedes se emplean larvicidas organofosforados (temefos), insecticidas organofosforados (malation y fenitrotion) y piretroides (permetrina y deltametrina). Puede haber resistencia. Los derivados vegetales son menos costosos y biodegradables, entre ellos el aceite de cetronela, que microencapsulado se preserva de la evaporación.

Arboviruses are transmitted by arthropods, including those responsible for the current pandemic: alphavirus (Chikungunya) and flaviviruses (dengue and Zika). Its importance increased in the Americas over the past 20 years. The main vectors are Aedes aegypti and A. albopictus. Dengue infection provides long lasting immunity against the specific serotype and temporary to the other three. Subsequent infection by another serotype determines more serious disease. There is a registered vaccine for dengue, Dengvaxia (Sanofi Pasteur). Other two (Butantan and Takeda) are in Phase III in 2016. Zika infection is usually asymptomatic or occurs with rash, conjunctivitis and not very high fever. There is no vaccine or specific treatment. It can be transmitted by parental, sexual and via blood transfusion. It has been associated with microcephaly. Chikungunya causes prolonged joint pain and persistent immune response. Two candidate vaccines are in Phase II. Dengue direct diagnosis is performed by virus isolation, RT-PCR and ELISA for NS1 antigen detection; indirect methods are ELISA-IgM (cross-reacting with other flavivirus), MAC-ELISA, and plaque neutralization. Zika is diagnosed by RT-PCR and virus isolation. Serological diagnosis cross-reacts with other flavivirus. For CHIKV culture, RT-PCR, MAC-ELISA and plaque neutralization are used. Against Aedes organophosphate larvicides (temephos), organophosphorus insecticides (malathion and fenitrothion) and pyrethroids (permethrin and deltamethrin) are usually employed. Resistance has been described to all these products. Vegetable derivatives are less expensive and biodegradable, including citronella oil, which microencapsulated can be preserved from evaporation.

Humans , Animals , Dengue/diagnosis , Dengue/prevention & control , Dengue/transmission , Chikungunya Fever/diagnosis , Chikungunya Fever/prevention & control , Chikungunya Fever/transmission , Americas/epidemiology , Enzyme-Linked Immunosorbent Assay , Viral Vaccines/therapeutic use , Chikungunya virus/immunology , Aedes/virology , Dengue Virus/immunology , Dengue Vaccines/therapeutic use , Zika Virus/immunology , Zika Virus Infection/transmission , Insect Vectors/physiology , Insecticides
Mem. Inst. Oswaldo Cruz ; 111(3): 161-167, Mar. 2016. tab, graf
Article in English | LILACS | ID: lil-777371


Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient’s clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

Female , Humans , Male , Cytokines/metabolism , Dengue Virus/immunology , Severe Dengue/immunology , Dengue Virus/classification , Dengue/immunology , Enzyme-Linked Immunosorbent Assay , Inflammation Mediators/metabolism , Interferon-gamma/blood , /blood , /blood , /blood , Mexico , Real-Time Polymerase Chain Reaction/methods , Serogroup , Statistics, Nonparametric , Severe Dengue/blood , Tumor Necrosis Factor-alpha/blood