Búsqueda de flavivirus en individuos con sospecha clínica de dengue y resultado negativo para el antígeno NS1 en Paraguay / Search of flavivirus in individuals with clinical suspicion of dengue and negative for the NS1 antigen in Paraguay

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.

Improvement of a RT-PCR assay for Yellow Fever virus genome detection

The aim of the present study was to describe an improved protocol of reverse transcription polymerase chain reaction (RT-PCR) for Yellow Fever virus genome detection. A strain of ribonucleic acid of Yellow Fever virus was submitted to the improved protocol of RT-PCR and the amplicons were visualized under ultraviolet transilluminator, purifed and sequenced. The nucleotide sequence obtained was compared with sequences available in GenBank using the tblastx tool. The amplicons produced by the strain of ribonucleic acid of Yellow Fever virus exhibited fragments of 400 and 800 base pairs and the consensus sequence exhibited a similarity of 100% with Yellow Fever virus sequences recorded in GenBank. The improved protocol described in this study allowed Yellow Fever virus genome detection and enabled the elimination of the nested-PCR step, which has been frequently associated with contamination. In addition, it reduced the time of reaction, the cost of reagents and the possibility of sample contamination. New methods of investigating these infections must be elaborated and a continuous vigilance of these viruses in their different vectors and hosts is required to avoid negative impacts on human health, tourism and trade.(AU)

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Introduction Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. Methods A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. Results Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. Conclusions Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil. .

Aspectos epidemiológicos da Febre do Oeste do Nilo / Epidemiological aspects of West Nile Fever

Desde sua introdução na América do Norte em 1999, mais de 27.500 casos humanos da infecção por West Nile virus (WNV) foram reportados nos Estados Unidos da América (EUA), resultando em mais de 1000 casos fatais. Recentemente, a disseminação do vírus para o hemisfério sul foi confirmada com a detecção de animais infectados pelo WNV em território sul-americano. A soropositividade para WNV em eqüídeos na Colômbia e Venezuela e o isolamento do vírus nestes animais na Argentina, reiteram a necessidade da manutenção do sistema de vigilância enzoótica para WNV em território brasileiro. Aspectos pertinentes à infecção, patogenia e epidemiologia do WNV são discutidos neste artigo.

Fusogenic peptide as diagnostic marker for detection of flaviviruses.

BACKGROUND: Dengue, Japanese encephalitis, West Nile encephalitis, yellow fever are the common flaviviral diseases associated with high morbidity and mortality. The initial symptoms of most of the flaviviral infections are similar to each other as well as to some other viral diseases. Making clinical diagnosis, therefore, becomes a challenging task for the clinician. Several studies have been reported on using detection of serum antibodies against flavivirus for the diagnosis of specific flaviviral disease; no field-based pan-flavi virus detection system is available, which can be used in low-endemicity areas for differentiation of flaviviral disease from other viral diseases. AIM: To identify a conserved amino acid sequence among all flaviviruses and evaluate the antibody formed against the conserved peptide to develop pan-flavivirus detection system. MATERIALS AND METHODS: In the present study we have compared amino acid sequences of several flaviviruses and identified a conserved amino acid sequence lying in domain II of envelope protein. RESULTS: A peptide having the conserved amino acid sequence was used to generate polyclonal antibodies and these antibodies were used to detect several flaviviruses. Anti-peptide polyclonal antibodies selectively recognized flaviviruses and did not detect non-flaviviruses. Anti-peptide antibodies detected presence of virus in serum spiked with pure virus preparations. CONCLUSION: The study offers a rationale for development of pan-flavivirus capture assay suitable for low endemic areas.

Limitaciones para el serodiagnóstico del virus del oeste del Nilo en zonas endémicas con co-circulación de Flavivirus en el Caribe colombiano / Limitations to West Nile Virus serodiagnosis in endemic zones with co-circulation of Flavivirus in the Colombian Caribbean coast

Objetivo: El propósito de este estudio fue el de establecer la presencia de anticuerpos IgG contra el VON en una población de la costa atlántica utilizando dos pruebas comerciales y establecer su utilidad. Materiales y método: Se analizaron de forma aleatoria 52 muestras de sueros de personas que trabajaban en labores agrícolas desde hacia más de 15 años en el departamento de Sucre. Se utilizó la técnica West Nile Virus IgG ELISA (Focus Technologies). También se utilizó la prueba de inmunofluorescencia (PANBIO Columbia. Arbovirus IgG-IFA slides), donde se evaluó la detección de anticuerpos contra cinco antígenos diferentes de arbovirus (encefalitis equina venezolana, encefalitis japonesa, fiebre amarilla y dengue). Resultados: Con la prueba de ELISA, de 52 sueros estudiados para la detección de anticuerpos contra VON, 38 (73/100) resultaron positivos, los 14 (27/100) restantes resultaron negativos. Con la prueba de IFA, 6 sueros (11.5/100) resultaron seropositivos débiles para VON, 46 (88.5/100) resultaron negativos para la detección de anticuerpos contra el VON. Utilizando IFA se presentaron reacciones cruzadas contra otros arbovirus como dengue, encefalitis venezolana y japonesa y fiebre amarilla. El estudio demostró la complejidad del serodiagnóstico de Flavivirus en las zonas endémicas como la del Caribe colombiano.Conclusión: Los resultados obtenidos en este estudio demuestran que las pruebas de ELISA e IFA para humanos son de poca utilidad diagnóstica contra el VON en las zonas colombianas donde se presentan circulación de otros Flavivirus como dengue, fiebre amarilla o encefalitis equina

Vírus do Nilo Ocidental: nova ameaça à segurança transfusional / West Nile virus: a new threat to transfusion safety

O vírus do Nilo Ocidental (West Nile Virus) é um RNA vírus, dos mais importantes flavivírus patogênicos em humanos. Devido ao número crescente de casos confirmados de infeção pelo vírus do Nilo Ocidental nos EUA a partir de 1999, com a documentação da sua disseminação da costa leste para a oeste e sul em um período inferior a três anos e pela sua disseminação em quatro continentes, medidas estão sendo implementadas para o controle dessa epidemia. A documentação da transmissão pela transfusão de sangue, órgãos transplantados, aleitamento materno e transmissão vertical e a observação de taxa significante de morbidade e mortalidade (variando de 4 por cento a 29 por cento) está alarmando a comunidade médica internacional. Esforços estão sendo realizados na tentativa de obtenção de testes diagnósticos precisos, na busca de uma terapêutica eficaz - uma vez que o meio de controle mais efetivo no momento é o controle de vetores (insetos) - e no desenvolvimento de vacinas. Tendo em vista a detecção de casos na América Central e pelas condições climáticas ideais do Brasil, devemos estar atentos quanto aos possíveis riscos dessa epidemia. Esse artigo apresenta o quadro atual mundial de disseminação, modos de transmissão, quadro clínico, diagnóstico e tratamento, e algumas medidas preventivas para o controle do vírus do Nilo Ocidental.

Prevalencia de anticuerpos contra flavivirus en jóvenes voluntarios ingresantes al servicio militar de la Provincia de Formosa, Argentina / Seropositive against Flavivirus in young recruits from the Province of Formosa, Argentina

El objetivo del presente trabajo fue conocer la prevalencia de anticuerpos en poblaciones de riesgo a los Flavivirus. Se analizaron 189 sueros humanos provenientes de 3 localidades de la Provincia de Formosa. La región estudiada fue seleccionada por su proximidad a Brasil y Paraguay con el fin de verificar la probable introducción de Flavivirus de estos pa1ses, especialmente dengue y fiebre amarilla o la emergencia de los ya existentes en nuestro país. Se realizaron las pruebas de inhibición de la hemoaglutinación (IH), fijación del Complemento (FC) y neutralización (NT), utilizando los virus de la encefalitis de San Luis (ESL), Bussuquara, Ilheus, fiebre amarilla (FA)y dengue subtipos 1 y 2. Todos los sueros fueron negativos por IH para dengue e Ilheus. Por esta prueba un suero fue positivo sólo para FA, y dos sólo para Bussuquara, confirmándose uno por NT. Un total de 22 sueros fue positivo para ESL por IH y 40 sueros reaccionaron por la prueba de NT contra el mismo virus. La prevalencia de anticuerpos IH y NT fue similar para las tres localidades estudiadas. Estos resultados muestran que el virus ESL circula efectivamente en la zona estudiada con un valor de prevalencia de anticuerpos IH y NT significativo y que el mencionado virus podría cumplir un rol importante en infecciones febriles de etiología viral no confirmados en esa zona de nuestro país