Genome analysis of yellow fever virus of the ongoing outbreak in Brazil reveals polymorphisms

The current yellow fever outbreak in Brazil is the most severe one in the country in recent times. It has rapidly spread to areas where YF virus (YFV) activity has not been observed for more than 70 years and vaccine coverage is almost null. Here, we sequenced the whole YFV genome of two naturally infected howler-monkeys (Alouatta clamitans) obtained from the Municipality of Domingos Martins, state of Espírito Santo, Brazil. These two ongoing-outbreak genome sequences are identical. They clustered in the 1E sub-clade (South America genotype I) along with the Brazilian and Venezuelan strains recently characterised from infections in humans and non-human primates that have been described in the last 20 years. However, we detected eight unique amino acid changes in the viral proteins, including the structural capsid protein (one change), and the components of the viral replicase complex, the NS3 (two changes) and NS5 (five changes) proteins, that could impact the capacity of viral infection in vertebrate and/or invertebrate hosts and spreading of the ongoing outbreak.

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)

Clave fotográfica para hembras de Haemagogus Williston 1896 (Diptera: Culicidae) de Venezuela, con nuevo registro para el país / Pictorial key for females of Haemagogus Williston 1896 (Diptera: Culicidae) from Venezuela, with a new record for the country

El género neotropical Haemagogus Williston, está representado por mosquitos de actividad diurna, cuyas fases inmaduras se crían en fitotelmatas (huecos de árboles e internodos cortados de bambú). Especies de este género se han señalado involucradas en la transmisión de la Fiebre Amarilla selvática, virus que circula en áreas boscosas de América Latina entre primates no humanos y marsupiales arborícolas por la picada de estos mosquitos. De las 28 especies reconocidas en el continente, 9 se encuentran en Venezuela. Una de ellas, Heamagogus (Conopostegus) clarki constituye un nuevo registro para el país. Se presenta una actualización de la taxonomía y de la distribución geográfica del género en Venezuela, así como la primera clave fotográfica con términos sencillos para el uso de personal no experimentado.

The neotropical genus Heamagogus Williston includes mosquitoes with diurnal activity and immature breeding on Phytotelmata (tree-holes and cut bamboo internodes). Haemagogus species have been involved in sylvatic yellow fever transmission, a virus circulating in forest areas in Latin America among arboreal primates and marsupials by means of mosquito bite. The genus comprises 28 species, nine of them occurring in Venezuela. One of these, Haemagogus (Comopostegus) clarki, is a new record for this country. We show here an update of the taxonomic status and the geographical distribution of the genus in Venezuela and the first photographical key using simple terms for non-expert personnel

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.

Yellow fever virus isolated from a fatal post vaccination event: an experimental comparative study with the 17DD vaccine strain in the Syrian hamster (Mesocricetus auratus) / Vírus da febre amarela isolado de acidente pós-vacinal fatal: estudo experimental comparativo com a amostra vacinal 17DD em Syrian hamsters (Mesocricetus auratus)

In order to investigate the pathogenicity of the virus strain GOI 4191 that was isolated from a fatal adverse event after yellow fever virus (YFV) vaccination, an experimental assay using hamsters (Mesocricetus auratus) as animal model and YFV 17DD vaccine strain as virus reference was accomplished. The two virus strains were inoculated by intracerebral, intrahepatic and subcutaneous routes. The levels of viremia, antibody response, and aminotransferases were determined in sera; while virus, antigen and histopathological changes were determined in the viscera. No viremia was detected for either strain following infection; the immune response was demonstrated to be more effective to strain GOI 4191; and no significant aminotransferase levels alterations were detected. Strain GOI 4191 was recovered only from the brain of animals inoculated by the IC route. Viral antigens were detected in liver and brain by immunohistochemical assay. Histothological changes in the viscera were characterized by inflammatory infiltrate, hepatocellular necrosis, and viral encephalitis. Histological alterations and detection of viral antigen were observed in the liver of animals inoculated by the intrahepatic route. These findings were similar for both strains used in the experiment; however, significant differences were observed from those results previously reported for wild type YFV strains.

Visando investigar a possível patogenicidade do vírus isolado (GOI 4191) de um evento adverso fatal pela vacinação antiamarílica, realizou-se um ensaio experimental em Syrian hamsters (Mesocricetus auratus), usando-se a cepa vacinal 17DD como parâmetro. As amostras virais foram inoculadas por via intracerebral, intra-hepática e subcutânea. Nos soros foram determinados níveis de viremia, resposta imune e aminotransferases, e nas vísceras a presença de vírus, antígeno e lesões teciduais. Não se detectou viremia para as duas amostras, a resposta imune foi maior para GOI 4191, e as aminotransferases não apresentaram alterações compatíveis com danos hepáticos. Nos animais inoculados por via intracerebral o vírus foi recuperado somente a partir do cérebro, sendo o antígeno viral detectado, por imuno-histoquímica, no cérebro e fígado. Infiltrado inflamatório e corpúsculos acidófilos foram observados no fígado e lesões tipo encefalite viral no sistema nervoso central. Alterações histológicas e antígeno viral foram observados, também, no fígado dos animais infectados por via intra-hepática, e ausentes naqueles inoculados por via subcutânea. Os resultados foram similares para as duas amostras testadas, entretanto distintos daqueles relatados na literatura para cepas silvestres do vírus amarílico.

The yellow fever 17D vaccine virus: molecular basis of viral attennuation and it use as an expression vector

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed.