Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP). The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.

A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado. Antí-genos citoplasmáticos endógenos, caracteristicamente codificados por vacinas de DNA plasmidial, são majoritariamente apresentados ao sistema imune através de moléculas do Complexo Maior de Histocompatibilidade de classe I - MHC I. A via de apresentação MHC I é mais associada à resposta celular citotóxica e, frequentemente, não elicita uma resposta humoral satisfatória. Uma das principais estratégias para direcionar antígenos codificados pelas vacinas de DNA para o compartimento MHC II é expressar estes antígenos dentro da Proteína de Associação à Membrana Lisossomal (LAMP). A proteína do envelope dos flavivirus é reconhecidamente a principal proteína de superfície viral e o principal alvo para anticorpos neutralizantes. Diferentes grupos têm demonstrado que a co-expressão das proteínas de membrana e do envelope dos flavivirus em células de mamíferos, fusionada com a porção carboxi-terminal de LAMP, é capaz de induzir níveis satisfatórios de anticorpos neutralizantes. Neste trabalho revisamos a estratégia de co-expressão da proteína do envelope dos flavivírus, como quimeras de LAMP, com o objetivo de desenvolver vacinas de DNA contra a febre do Oeste do Nilo, dengue e febre amarela.

Dengue-yellow fever sera cross-reactivity; challenges for diagnosis / Reacción cruzada en sueros de Fiebre Amarilla y Dengue: desafíos para el diagnóstico

Objective The Flavivirus genera share epitopes inducing cross-reactive antibodies leading to great difficulty in differentially diagnosing flaviviral infections. This work was aimed at evaluating the complexity of dengue and yellow fever serological differential diagnosis. Material and methods Dengue antibody capture ELISA and a yellow fever neutralisation test were carried out on 13 serum samples obtained from yellow fever patients, 20 acute serum samples from dengue patients and 19 voluntan/ serum samples pre- and post-vaccination with YF vaccine. Results Dengue ELISA revealed IgM reactivity in 46,2 percent of yellow fever patients and 42 percent of vaccinees. Sixteen out of 20 dengue patients (80 percent) had high YF virus neutralisation titres. Conclusions . Such very high cross-reactivity data challenged differential laboratory diagnosis of dengue and yellow fever in áreas where both flaviviruses co-circulate. New laboratory strategies are thus needed for improving the tests and providing a specific laboratory diagnosis. Cross-reactivity between Flaviviruses represents a great difficulty for epidemiological surveillance and preventing dengue, both of which demand urgent attention.

Objetivo Los miembros del genero Flavivirus poseen epítopes inductores de anticuerpos de reactividad cruzada, lo que representa una gran dificultad en el diagnostico diferencial. En este trabajo nos propusimos evaluar la complejidad del diagnostico diferencial entre dengue y fiebre amarilla. Materiales y métodos Ensayos de ELISA de captura para dengue y de neutralización para fiebre amarilla fueron realizados en 13 muestras de suero de pacientes con fiebre amarilla, 20 muestras de pacientes con dengue en fase aguda y 19 voluntarios sanos antes y después de ser vacunados para la fiebre amarilla. Resultados Los ensayos de ELISA para IgM contra dengue mostraron reactividad cruzada en el 46,2 por ciento de los pacientes con fiebre amarilla y 42 por ciento de los vacunados. Dieciséis de los 20 pacientes con Dengue (80 por ciento) tuvieron altos títulos de anticuerpos neutralizantes para fiebre amarilla. Conclusión La alta reactividad cruzada que se encontró, representa un desafío para el diagnostico diferencial del dengue y fiebre amarilla en áreas donde co-circulan estos Flavivirus. Nuevas estrategias de diagnóstico de laboratorio son necesarias para suministrar un diagnóstico especifico. La reactividad cruzada entre Flavivirus representa un gran problema para la vigilancia epidemiológica, control y prevención del dengue, el cual debe ser solucionado.

Genes de susceptibilidad/resistencia a Flavivirus, implicaciones en la severidad de la infeccion / Susceptibility/Resistance Genes to Flavivirus, Implications on Disease Severity

Las infecciones transmitidas por Flavivirus se encuentran entre las enfermedades transmisibles con mayor incidencia en el mundo. La mayoría de ellas se manifiestan clínicamente como un síndrome febril que puede estar o no acompañado de diversos síntomas. La severidad de estas infecciones es variable con casos asintomáticos y otros que pueden llegar a ser letales. La razón de esta variabilidad en la presentación clínica, se desconoce en humanos. En ratones se han identificado cepas susceptibles y cepas resistentes a la infección por algunos Flavivirus. Por clonación posicional se mapeó el gen responsable de la resistencia a virus West Nile en el cromosoma 5 de ratón y se identificó como oligoadenilato sintetasa 1b (Oas1b). Este gen codifica una proteína que sintetiza oligómeros de adenina que activan la RNasaL, que a su vez degrada los RNAs virales. Células provenientes de ratones resistentes a la infección por Flavivirus producen menor cantidad de virus que su contraparte susceptible. Recientemente en humanos, se identificó un polimorfismo asociado con susceptibilidad a infección por virus West Nile en el gen de OasL. Sin embargo, el mecanismo bioquímico y molecular exacto por el cual se produce la susceptibilidad no ha sido completamente dilucidado. Este conocimiento permitiría aclarar aspectos de la fisiopatología de estas enfermedades y enfocar la terapéutica desde un punto de vista más específico.

Diagnóstico sorológico de infecções por dengue e febre amarela em casos suspeitos no Estado do Pará, Brasil, 1999 / Serological diagnosis of dengue and yellow fever infections in suspected cases from Pará State, Brazil, 1999

De junho a dezembro de 1999, foram coletadas 785 amostras de soro de pacientes com suspeita clínica de dengue e/ou febre amarela. Os pacientes foram atendidos nas unidades de saúde distribuídas pelas seis mesorregiöes do Estado do Pará, Brasil. As amostras de soro foram testadas pelo método de inibiçäo da hemaglutinaçäo para detecçäo de anticorpos para Flavivirus e pelo ensaio imunoenzimático para detecçäo de imunoglobulina M para dengue e febre amarela. Das amostras coletadas, 563 (71,7 por cento) foram positivas pelo IH, e dentre estas 150 (26,6 por cento) foram positivas pelo ELISA-IgM. O vírus dengue foi responsável pela maioria das infecçöes recentes em todas as mesorregiöes e os casos de febre amarela detectados neste estudo foram restritos às mesorregiöes Marajó e Sudeste

The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain

Antigenic relatedness of selected flaviviruses: study with homologous and heterologous immune mouse ascitic fluids

A relacao antigenica de 9 Flavivirus, febre amarela (YF), Wesselsbron (WSL), Uganda S (UGS), Potiskum (POT), West Nile (WN), Banzi (BAN), Zika (ZK), Dengue tipo 1 (DEN-1) e Dengue tipo 2 (DEN-2), foi avaliada por reacao de inibicao da hemaglutinacao cruzada (cross-HI) e reacao de fixacao do complemento cruzada (Cross-CF) entre cada um dos virus e seu fluido ascitico homologo em camundongos. Medias de titulos foram calculadas usando os titulos heterologos e homologos. Reacoes cruzadas CF revelaram maiores variacoes antigenicas entre virus do que reacoes cruzadas HI. Nao houve variacao antigenica significativa entre virus WSL, POT e YF usando cada um dos metodos. Todavia, diferencas definidas da antigenicidade foram observadas entre eles e os virus UGS, BAN e ZK. Nao existiram diferencas significativas entre UGS, BANe ZK ou entre DEN-1 e DEN-2. A relacao sorologica entre Flavivirus e importante para se estabelecer o diagnostico e a epidemiologia destas infeccoes na Africa

Response to JE vaccine among HIV-infected children, Bangkok, Thailand.

Since 1990, Japanese encephalitis (JE) vaccine has been part of EPI in northern Thailand, where there is a high prevalence of JE and HIV infection. To evaluate the immunogenicity and safety of JE vaccine among HIV-infected children, we conducted a retrospective study of HIV-infected and uninfected children who received 2 doses of JE vaccine at 12 months of age. Pre- and post-immunization plasma specimens were tested by plaque reduction neutralization for antibody levels to JE and dengue(1-4) viruses; titers of > or =10 were considered positive. Excluding 5 children with preimmunization antibodies, 5 of 14 (36%) HIV-infected children and 18 of 27 (67%) uninfected children had positive JE antibody titers after immunization [odds ratio (OR) 0.3, p=0.06]; 31% absolute difference [95% confidence interval (CI) 0-61.7%). The geometric mean titer of HIV-infected children with positive titers was lower than that of control children (15.1 vs, 23.8; p=0.17). No significant vaccine-associated adverse events were noted. We conclude that primary antibody response to JE vaccine was low among HIV-infected children and was approximately half of that seen among uninfected children. In endemic areas, HIV-infected children are likely to be at risk of acquiring JE despite routine immunization with 2 doses.

Molecular approach to the development of flavivirus vaccines

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members present in most continents. Among the most important are yellow fever (YF), dengue (DEN) with its 4 serotypes and Japanese escephalitis (JE) virus. A live attenuated virus is used as a cost-effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The development of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. The new approaches include the use of cDNAs encompassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated virues. The use of infectious cDNA as a carrier for heterologous antigens is a gaining importance since chimeric viruses are shown to be viable, immunogenic and less virulent in some cases as compared to the parental viruses. The use of DNA to overcome intrinsic mutation rates of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost of live attenuated vaccines.

O uso do teste imunoenzimático na vigilância epidemiologica de arboviroses / The use of enzyme-linked immunosolvent assay in the epidemiologic surveillance of arbovirus

Realizou-se revisäo bibliográfica sobre a utilizaçäo do teste imunoenzimático, ELISA na vigilância Epidemiológica de infecçöes causadas por arbovírus da família Flaviviridae, gênero Flavivirus e da Família Togaviridae, gênero Alphavirus. O teste tem sido empregado na pesquisa de anticorpos humanos, de anticorpos e antígenos em reservatórios näo humanos e na identificaçäo de antígenos e da fonte alimentar de mosquitos vetores. Analisou-se o desempenho de ELISA comparando-o a técnicas empregadas para identificaçäo de anticorpos e antígenos de arbovirus. O teste apresentou 100,0 por cento de sensibilidade e especificidade média de 84,5 por cento na identificaçäo de anticorpos anti-Alphavirus em humanos. Foi muito sensível para Flavivirus, com valor médio de 95,2 por cento e especificidade média de 77,6 por cento. Na identificaçäo de anticorpos anti-arbovirus em reservatórios näo humanos, ELISA mostrou sensibilidade de 100,0 por cento e especificidade de 97,4 por cento e na pesquisa de antígenos virais em mosquitos vetores, especificidade média de 93,6 por cento e sensibilidade média de 76,5 por cento. Apresentou alto valor preditivo positivo, observado no cálculo da média dos valores apresentados nos trabalhos em que esse parâmetro foi pesquisado e obteve-se um resultado de 89,0 por cento. No estudo da reprodutividade do teste, observou-se coeficiente de variaçäo de 3,0 a 14,0 por cento nos resultados

Ocorrência recente de infecçäo humana por arbovírus Rocio na regiäo do Vale do Ribeira / Recent occurrence of human infection by Rocio arbovirus in Ribeira Valley, Brazil

Descreve-se o achado sorológico de anticorpos IgM para vírus Rocio em duas crianças da área rural do Vale do Ribeira, Estado de Säo Paulo. O vírus Rocio foi responsável por extensa epidemia de encefalite na regiäo na regiäo em 1975-1977. Desde 1980 näo têm sido diagnosticados casos de doença. Impöe-se a ativaçäo de medidas dirigidas a vigilância epidemiológica desta arbovirose

Localization of arboviral antigen in brain tissues from patients with encephalitis.

Brain tissues from 38 patients with a clinical suspicion of encephalitis or encephalopathy were examined by two immunoenzymatic techniques for the detection of arboviral antigen. Group B arboviral antigen was identified in 23 of these tissues. This simple method could be used for the diagnosis of the causal agent of encephalitis.

The neurovirulence of flaviviruses in crab-eating monkeys (Macaca fascicularis).

The neurovirulent properties of attenuated dengue-2 and yellow fever (YF) vaccines, dengue-2 (DEN-2) and Japanese encephalitis (JE) viruses were studied in crab-eating monkeys (Macaca fascicularis). Number of central nervous system sites (as proportion affected) with neurovirulence (NV) lesions were compared. The results indicate that these monkeys reliably developed NV-lesion when inoculated with either JE or YF vaccine viruses (87%). NV-lesions occurred in a minority when inoculated with DEN-2 vaccine virus, were of minimal severity (9%), were probably biologically insignificant, and were of equal or less severity than lesions produced by its parental virus (10%).

Programa e Resumos do 21§ Congresso da Sociedade Brasileira de Medicina Tropical

Soros de 75 pacientes que apresentaram encefalite, foram testados por técnica imunoenzimática, ELISA, utilizando-se microplacas de poliestireno sensibilizadas com anti-IgM humana. Dos 75 doentes, 23 apresentavam sorologia positiva indicativa de infecçäo aguda por vírus Rocio, 48 tinham resultados sorológicos inconclusivos e 4, soros näo reagentes para o vírus. No desenvolvimento do teste imunoenzimático as placas foram incubadas com os soros dos pacientes nas diluiçöes 1/10 e 1/100, com os extratos antigênicos dos flavivírus Rocio, Ilhéus e da encefalite St. Louis, préviamente titulados, e com o conjugado de anticorpo monoclonal marcado com fosfatase alcalina. A reaçäo foi revelada com substrato de p-nitrofenil fosfato e avaliada espectrofotométricamente em 410nm; nos testes foram incluídos controles de soros negativos e positivo e conjugado. Obtiveram-se resultados positivos com teste ELISA nos 23 pacientes com diagnóstico confirmado (nas 2 amostras de soro em 21 e em uma amostra em 2) e em 85,4% (41/48) dos doentes com resultados inconclusivos por IH e FC. Nos 4 soros näo reagentes o teste ELISA foi negativo. Para verificar a possível presença de anticorpos IgM näo específicos, 35 soros positivos por ELISA foram retestados depois de absorvidos com gama-globulina agregada pelo calor,segundo procedimentos de Camargo et al. (1972), observando-se em 34 variaçäo näo significativa dos valores obtidos antes da absorçäo

Flavivirus: encuesta serológica en equinos del partido de Tandil / Flavivirus: serological survey in horses from Tandil county

Se obtivieron 282 sueros de equinos cuyas edades oscilan entre 45 días y 27 años provenientes del partido de Tandil y alrededores para investigar anticuerpos contra 3 flavivirus: Ilheus (ILH), Encefalitis de San Luis (SLE) y Fiebre amarilla (YF) mediante las técnicas de inhibición de la hemaglutinación (IH), fijación de complemento (FC) y neutralización (NT). Las muestras se recolectaron entre el 20-3-79 y el 25-11-80. Cuarenta y nueve sueron reaccionaron positivamente contra uno o más de los flavivirus usados por IH y/o FC, representando una prevalencia del 17,4% para este complemento antigénico. Veinticuartro de ellos neutralizaron ILH, 6 SLE, y de ellos, 2 neutralizaron simultáneamente a ambos virus. Veintiún sueros no neutralizaron ni ILH ni SLE, por cual no podemos atribuir los anticuerpos específicamente a estos virus, sino a alguno/s con ellos relacionados, que podrían corresponder a flavivirus presentes en el medio ambiente pero no en nuestras pruebas. Otros 12 sueron negativos para los tres antígenos por IH y/o FC tampoco neutralizaron SLE o ILH. Los resultaods muestran que ILH y SLE, ambos virus aislados años atrás en Argentina, son endémicos en el área bajo estudio y que los mismos infectan a equinos, quinzás sin oroducirles enfermedades detectables, ya que no han sido reportadas epizzotias equinas en la población investigada, atribuidas a SLE o ILH