The relevance of dengue virus genotypes surveillance at country level before vaccine approval.

Dengue is a major threat for public health in tropical and subtropical countries around the world. In the absence of a licensed vaccine and effective antiviral therapies, control measures have been based on education activities and vector elimination. Current efforts for developing a vaccine are both promising and troubling. At the advent of the introduction of a tetravalent dengue vaccine, molecular surveillance of the circulating genotypes in different geographical regions has gained considerable importance. A growing body of in vitro, preclinical, and clinical phase studies suggest that vaccine conferred protection in a geographical area could depends on the coincidence of the dengue virus genotypes included in the vaccine and those circulating. In this review we present the state-of-the-art in this field, highlighting the need of deeper knowledge on neutralizing immune response for making decisions about future vaccine approval and the potential need for different vaccine composition for regional administration.

Desarrollo de un método de transcripción inversa seguida de reacción en cadena de la polimerasa para la detección del virus de la fiebre amarilla / Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection

Introduction. Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. Objective. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. Materials and methods. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. Results. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. Conclusion. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Introducción. La fiebre amarilla se considera una enfermedad reemergente y endémica en regiones tropicales de África y Suramérica. Actualmente, no existen estuches estandarizados o comerciales disponibles para la detección del virus de la fiebre amarilla y, por lo tanto, el diagnóstico debe hacerse mediante técnicas de rutina que consumen mucho tiempo y algunas veces no garantizan la detección del virus o de sus proteínas. Además, la cocirculación con otros flavivirus, incluyendo el del dengue, hacen el diagnóstico más complicado. Objetivo. Desarrollar un ensayo específico de amplificación basado en transcripción inversa seguida de reacción en cadena de la polimerasa, con el fin de mejorar la detección y el diagnóstico de la fiebre amarilla, tanto a partir de suero como de tejido fresco. Materiales y métodos. Se diseñaron iniciadores específicos para amplificar un fragmento conservado del virus de la fiebre amarilla. Un segundo par de iniciadores se usó en una reacción de amplificación anidada para incrementar la sensibilidad. Se probaron 33 muestras clínicas con la técnica estandarizada. Resultados. El amplímero esperado se obtuvo en 25 de las 33 muestras analizadas usando este método y 2 más resultaron positivas después de la reacción anidada. Conclusión. Esta técnica mejorada garantiza la detección de todos los genotipos virales de fiebre amarilla y puede incrementar la sensibilidad del ensayo introduciendo una segunda etapa de amplificación, lo cual permite el diagnóstico diferencial con infección por dengue y otros flavivirus, lo cual es de gran importancia para la vigilancia y la toma de medidas epidemiológicas oportunas.

Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

INTRODUCTION: Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. OBJECTIVE: To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. MATERIALS AND METHODS: RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. RESULTS: The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. CONCLUSION: This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Advanced yellow fever virus genome detection in point-of-care facilities and reference laboratories.

Reported methods for the detection of the yellow fever viral genome are beset by limitations in sensitivity, specificity, strain detection spectra, and suitability to laboratories with simple infrastructure in areas of endemicity. We describe the development of two different approaches affording sensitive and specific detection of the yellow fever genome: a real-time reverse transcription-quantitative PCR (RT-qPCR) and an isothermal protocol employing the same primer-probe set but based on helicase-dependent amplification technology (RT-tHDA). Both assays were evaluated using yellow fever cell culture supernatants as well as spiked and clinical samples. We demonstrate reliable detection by both assays of different strains of yellow fever virus with improved sensitivity and specificity. The RT-qPCR assay is a powerful tool for reference or diagnostic laboratories with real-time PCR capability, while the isothermal RT-tHDA assay represents a useful alternative to earlier amplification techniques for the molecular diagnosis of yellow fever by field or point-of-care laboratories.

First international external quality assessment study on molecular and serological methods for yellow fever diagnosis.

OBJECTIVE: We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. STUDY DESIGN: For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. RESULTS: Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. CONCLUSION: This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.

Phylogenetic reconstruction of dengue virus type 2 in Colombia.

BACKGROUND: Dengue fever is perhaps the most important viral re-emergent disease especially in tropical and sub-tropical countries, affecting about 50 million people around the world yearly. In Colombia, dengue virus was first detected in 1971 and still remains as a major public health issue. Although four viral serotypes have been recurrently identified, dengue virus type 2 (DENV-2) has been involved in the most important outbreaks during the last 20 years, including 2010 when the fatality rate highly increased. As there are no major studies reviewing virus origin and genotype distribution in this country, the present study attempts to reconstruct the phylogenetic history of DENV-2 using a sequence analysis from a 224 bp PCR-amplified product corresponding to the carboxyl terminus of the envelope (E) gene from 48 Colombian isolates. RESULTS: As expected, the oldest isolates belonged to the American genotype (subtype V), but the strains collected since 1990 represent the American/Asian genotype (subtype IIIb) as previously reported in different American countries. Interestingly, the introduction of this genotype coincides with the first report of dengue hemorrhagic fever in Colombia at the end of 1989 and the increase of cases during the next years. CONCLUSION: After replacement of the American genotype, several lineages of American/Asian subtype have rapidly spread all over the country evolving in new clades. Nevertheless, the direct association of these new variants in the raise of lethality rate observed during the last outbreak has to be demonstrated.

Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia.

BACKGROUND: Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70's when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution. RESULTS: We used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major amino acid changes in the analyzed region were found. CONCLUSION: DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages.

Simultaneous circulation of genotypes I and III of dengue virus 3 in Colombia.

BACKGROUND: Dengue is a major health problem in tropical and subtropical regions. In Colombia, dengue viruses (DENV) cause about 50,000 cases annually, 10% of which involve Dengue Haemorrhagic Fever/Dengue Shock Syndrome. The picture is similar in other surrounding countries in the Americas, with recent outbreaks of severe disease, mostly associated with DENV serotype 3, strains of the Indian genotype, introduced into the Americas in 1994. RESULTS: The analysis of the 3'end (224 bp) of the envelope gene from 32 DENV-3 strains recently recovered in Colombia confirms the circulation of the Indian genotype, and surprisingly the co-circulation of an Asian-Pacific genotype only recently described in the Americas. CONCLUSION: These results have important implications for epidemiology and surveillance of DENV infection in Central and South America. Molecular surveillance of the DENV genotypes infecting humans could be a very valuable tool for controlling/mitigating the impact of the DENV infection.

Detección por reacción en cadena de la polimerasa de transcriptasa inversa del virus de la fiebre amarilla en monos silvestres: una herramienta sensible para la vigilancia epidemiológica / Detection of yellow fever virus by reverse transcriptase polymerase chain reaction in wild monkeys: a sensitive tool for epidemiologic surveillance

Introducción. La fiebre amarilla es una enfermedad zoonótica mantenida en la naturaleza por primates no humanos; su vigilancia por técnicas sensibles de laboratorio es necesaria para hacer evidente la actividad viral en territorio selvático. Objetivo. Detectar el virus de la fiebre amarilla en muestras de tejido hepático de primates no humanos, mediante la técnica de reacción en cadena de la polimerasa de transcriptasa inversa (RT-PCR) con iniciadores diagnósticos específicos. Materiales y métodos. Se procesaron muestras de tejido hepático de cinco monos del genero Alouatta spp. encontrados muertos en territorio selvático de los departamentos de Cesar y Magdalena entre diciembre de 2003 y junio de 2004. Las muestras fueron tratadas con una solución de lisis para aislar el ARN viral que, posteriormente, fue utilizado en una RT-PCR, utilizando iniciadores específicos para fiebre amarilla; paralelamente, se identificaron proteínas virales mediante inmunohistoquímica sobre cortes de tejido hepático incluidos en parafina. Resultados. Se obtuvieron productos de amplificación del tamaño esperado, (424 pb) en cuatro de las muestras analizadas; estas muestras mostraron, además, una reacción inmunohistoquímica positiva, lo que confirma la presencia del virus. Conclusión. El hallazgo del virus de la fiebre amarilla en monos silvestres representa una evidencia de su actividad enzoótica en nuestro territorio, que incrementa el riesgo de transmisión a humanos y de urbanización por procesos de migración de la población. La detección por técnicas moleculares rápidas y específicas del virus en monos silvestres representa una herramienta de vigilancia epidemiológica que permite activar de manera precoz los sistemas de control necesarios para impedir brotes y epidemias.

Introduction. Yellow fever is a zoonotic infection maintained in nature by non-human primates. Appropriate surveillance with sensitive laboratory techniques is necessary to evidence viral activity in the tropical forest habitats of these primates. Objective. Yellow fever virus was detected in hepatic tissue samples from non-human primates by reverse transcriptase polymerase chain reaction (RT-PCR) technique using specific primers for diagnosis. Materials and methods. Hepatic tissue samples were processed from five monkeys belonging genus Alouatta spp found dead in sylvatic areas of Cesar and Magdalena Provinces, Colombia, between December 2003 and June 2004. Samples were treated with lysis buffer prior to the isolation of viral RNA, which was then subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using yellow fever-specific primers. Simultaneously, viral proteins were identified by immunohistochemistry on parafin-embedded hepatic tissue. Results. The PCR method amplified fragments of the expected size (424 bp) in four of the tested samples. In addition, these samples showed a positive reaction by immunohistochemistry, supporting the evidence that the virus was present. Conclusion. The detection of yellow fever virus in wild monkeys was clear evidence of enzootic activity in northern Colombia. Increased probability of yellow fever transmission among human populations is indicated due to urbanization processes as a consequence of forced migration and displacement of the human populations. Molecular tests for rapid and specific detection of yellow fever in tissue samples of non-human primates is an important tool for epidemiologic surveillance. Rapid virus identification will permit the timely activation of control systems for prevention of further cases and epidemic situations.

[Detection of yellow fever virus by reverse transcriptase polymerase chain reaction in wild monkeys: a sensitive tool for epidemiologic surveillance]. / Detección por reacción en cadena de la polimerasa de transcriptasa inversa del virus de la fiebre amarilla en monos silvestres: una herramienta sensible para la vigilancia epidemiológica.

INTRODUCTION: Yellow fever is a zoonotic infection maintained in nature by non-human primates. Appropriate surveillance with sensitive laboratory techniques is necessary to evidence viral activity in the tropical forest habitats of these primates. OBJECTIVE: Yellow fever virus was detected in hepatic tissue samples from non-human primates by reverse transcriptase polymerase chain reaction (RT-PCR) technique using specific primers for diagnosis. MATERIALS AND METHODS: Hepatic tissue samples were processed from five monkeys belonging genus Alouatta spp found dead in sylvatic areas of Cesar and Magdalena Provinces, Colombia, between December 2003 and June 2004. Samples were treated with lysis buffer prior to the isolation of viral RNA, which was then subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using yellow fever-specific primers. Simultaneously, viral proteins were identified by immunohistochemistry on parafin-embedded hepatic tissue. RESULTS: The PCR method amplified fragments of the expected size (424 bp) in four of the tested samples. In addition, these samples showed a positive reaction by immunohistochemistry, supporting the evidence that the virus was present. CONCLUSION: The detection of yellow fever virus in wild monkeys was clear evidence of enzootic activity in northern Colombia. Increased probability of yellow fever transmission among human populations is indicated due to urbanization processes as a consequence of forced migration and displacement of the human populations. Molecular tests for rapid and specific detection of yellow fever in tissue samples of non-human primates is an important tool for epidemiologic surveillance. Rapid virus identification will permit the timely activation of control systems for prevention of further cases and epidemic situations.

[Molecular detection of yellow fever virus in human sera and mice brains]. / Detección molecular del virus de la fiebre amarilla en muestras de suero de casos fatales humanos y en cerebros de ratón.

A molecular method for the diagnosis of yellow fever virus infection was developed based on reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification. Examinations were conducted on lyophilized sera from 3 fatal yellow fever cases and 4 fresh sera from 3 fatal cases and one from a symptomatic patient (positive IgM against yellow fever virus). Sera were extracted with TRIZOL-LS to isolate viral RNA for RT treatment and the PCR reaction included 2 primers sets designed specifically for yellow fever virus: sense, JM2104 (5'-CGTTGGGAGAGGAGATTC-3') y JM2249 (5'-TTCTTCACTTCGGTTGGG-3'), and antisense, JM2673 (5'-TCATCTGCCCTGCTTCTC-3') y JM2751 (5'-CCTCTCTGGTAAACATTCT-3'). The technique for demonstrating the yellow fever virus in tissue samples was used in infected mice brains treated with lysis buffer before RNA extraction. PCR reactions were evaluated in agarose gels where single bands of the expected size for each primers pair (569 bp and 502 bp) were observed for all serum samples. In addition, the results for 2 fresh positive sera were supported by histopathologic finding of yellow fever virus. The RT-PCR method permits a rapid and specific demonstration of the presence of yellow fever virus.

Detección molecular del virus de la fiebre amarilla en muestras de suero de casos fatales humanos y en cerebros de ratón / Molecular detection of yellow fever virus in human sera and mice brains

Hemos adaptado un método molecular basado en la técnica de transcripción reversa seguida de la reacción en cadena de la polimerasa (RT-PCR) para diagnóstico alternativo de la infección por el virus de la fiebre amarilla. Se tomaron tres sueros liofilizados de casos fatales de fiebre amarilla y cuatro sueros frescos, de los cuales tres pertenecían a casos fatales de la enfermedad y el cuarto a un paciente sintomático con serología IgM positiva para fiebre amarilla; los sueros fueron tratados con Trizol-LS( para extraer el ARN viral que fue sometido a reacción de RT y posteriormente a PCR, para la cual se diseñaron dos parejas de iniciadores específicos de fiebre amarilla: iniciadores directos (sentido) JM2104 (5ï-CGTTGGGAGAGGAGATTC-3ï) y JM2249 (5ï-TTCTTCACTTCGGTTGGG-3ï), e iniciadores inversos (antisentido) JM2673 (5ï-TCATCTGCCCTGCTTCTC-3ï) y JM2751 (5ï-CCTCTCTGGTAAACATTCT-3ï). La aplicación de la técnica en tejidos se hizo en cerebros de ratón infectados con el virus amarílico, tratados con una solución de lisis antes de purificar el ARN. En geles de agarosa se observaron bandas únicas de amplificación del tamaño esperado (569 pb y 502 pb); todas las muestras fueron corroboradas con las dos parejas de iniciadores y en dos de las muestras de suero fresco los resultados positivos para fiebre amarilla fueron comprobados con estudio histopatológico. Este método de detección molecular permitió demostrar de manera r pida y eficiente la presencia del virus de la fiebre amarilla, hecho que tiene importantes implicaciones diagnósticas para este problema de salud pública.

Identification, cloning, and sequencing of different cytokine genes in four species of owl monkey.

Non-human primates could prove to be suitable models for the study of infectious diseases such as malaria, tuberculosis, and hepatitis; the molecules of their immune systems are in the process of being fully characterized. Due to the relevance of cytokines in the modulation of the immune response, a molecular analysis of these proteins in non-human primates from the Aotus genus was carried out. Peripheral blood mononuclear cells from four species of Aotusmonkey were obtained and their mRNAs for interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-gamma (IFN), and tumor necrosis factor (TNF)-alpha were characterized. This study shows a high degree of conservation between nucleotide and amino acid sequences of cytokines from different Aotus species and those from humans. The TNF-alpha molecules were identical in amino acid sequences for both.