Serological and molecular investigation of viral agents in freeliving jaguars of the Pantanal wetlands, state of Mato Grosso, Brazil / Investigação sorológica e molecular de agentes virais em onças-pintadas de vida livre no Pantanal de Mato Grosso, Brasil

This study investigates the exposure of free-living jaguars from two federal protected areas in the Pantanal of Mato Grosso, Brazil, to a variety viral agents. These viral agents, particularly causing zoonotic diseases, were analyzed using serological and molecular methods. None of the jaguars was positive by RT-PCR for the molecular detection of avian influenza and West Nile Fever (WNF). Only one animal was serologically positive for Eastern Equine Encephalitis (EEE) by virus neutralization test in VERO cell cultures, representing the first reported case of jaguar exposure to EEE virus. However, all the animals were negative for Western Equine Encephalitis (WEE) virus and Venezuelan Equine Encephalitis (VEE) virus. Eleven jaguars were tested by two tests for the detection of antibodies against rabies virus (Simplified Fluorescent Inhibition Microtest ­ SFIMT and Rapid Fluorescent Focus Inhibition Test ­ RFFIT), resulting in five positive animals, two animals in each test and one in both serological tests. Furthermore, three out of 14 samples subjected to the neutralization test were positive for antibodies against canine distemper virus (CDV), and 15 out of 17 samples subjected to the hemagglutination-inhibition test (HI) were positive for antibodies against canine parvovirus (CPV). In view of the findings of this study, it is unlikely that the viruses examined here represent a threat to the jaguar populations in this region.(AU)

Este estudo investigou a exposição de onças-pintadas de vida livre a agentes virais selecionados em duas unidades de conservação federais no Pantanal de Mato Grosso, Brasil. Para a análise desses agentes virais, a maioria de caráter zoonótico, foram utilizados métodos sorológicos e moleculares. Nenhuma das onze onças-pintadas examinadas foi positiva na técnica de real-time RT-PCR para a detecção molecular dos agentes da Influenza aviária e Febre do Nilo Ocidental (WNF). Somente um animal foi positivo sorologicamente para a o vírus da Encefalite Equina do Leste (EEE) pela Microtécnica de vírus neutralização em culturas de células VERO, sendo este o primeiro relato da exposição de onças-pintadas. Todos os animais examinados s foram negativos para o vírus da Encefalite Equina do Oeste (WEE) e Venezuelana (VEE). Amostras de soro colhidas de 11 onças-pintadas foram submetidas a adois testes distintos para a detecção de anticorpos contra o vírus da raiva (Teste Rápido de Inibição de Foco de Fluorescência ­ RFFIT e Microteste Simplificado de Inibição da Fluorescência - SFIMT), resultando em cinco animais positivos, dos quase dois positivos para cada teste e um positivo quando submetido aos dois testes sorológicos. Além disso, três das 14 amostras submetidas a técnica de soroneutralização foram positivas para a pesquisa de anticorpos contra o vírus da cinomose (CDV) e 15 amostras positivas das 17 analisadas para a pesquisa de anticorpos contra o parvovírus canino (CPV) foram identificadas pela técnica de Inibição da Hemaglutinação (HI). De acordo com os resultados deste estudo, é pouco provável que os agentes virais aqui analisados representem ameaça à população de onçaspintadas nesta região.(AU)

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the viruss glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.

Synergic effects between ocellatin-F1 and bufotenine on the inhibition of BHK-21 cellular infection by the rabies virus

Background Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal progressive acute encephalitis caused by a lyssavirus. Animal venoms and secretions have long been studied as new bioactive molecular sources, presenting a wide spectrum of biological effects, including new antiviral agents. Bufotenine, for instance, is an alkaloid isolated from the skin secretion of the anuran Rhinella jimi that inhibits cellular penetration by the rabies virus. Antimicrobial peptides, such as ocellatin-P1 and ocellatin-F1, are present in the skin secretion of anurans from the genus Leptodactylus and provide chemical defense against predators and microorganisms. Methods Skin secretion from captive Leptodactylus labyrinthicus was collected by mechanical stimulation, analyzed by liquid chromatography and mass spectrometry, and assayed for antiviral and cytotoxic activities. Synthetic peptides were obtained using solid phase peptide synthesis, purified by liquid chromatography and structurally characterized by mass spectrometry, and assayed in the same models. Cytotoxicity assays based on changes in cellular morphology were performed using baby hamster kidney (BHK-21) cells. Fixed Rabies virus (Pasteur Virus - PV) strain was used for virological assays based on rapid fluorescent focus inhibition test. Results Herein, we describe a synergic effect between ocellatin-F1 and bufotenine. This synergism was observed when screening the L. labyrinthicus skin secretion for antiviral activities. The active fraction major component was the antimicrobial peptide ocellatin-F1. Nevertheless, when the pure synthetic peptide was assayed, little antiviral activity was detectable. In-depth analyses of the active fraction revealed the presence of residual alkaloids together with ocellatin-F1. By adding sub-effective doses (e.g. < IC50) of pure bufotenine to synthetic ocellatin-F1, the antiviral effect was regained. Moreover, a tetrapetide derived from ocellatin-F1, based on alignment with the virus's glycoprotein region inferred as a possible cell ligand, was able to maintain the synergic antiviral activity displayed by the full peptide. Conclusions This novel antiviral synergic effect between a peptide and an alkaloid may present an innovative lead for the study of new antiviral drugs.(AU)

Equivalência e avaliação da necessidade de sorologia de controle entre esquemas de pré-exposição à raiva humana / Equivalencia y evaluación de la necesidad de serología de control entre esquemas de pre-exposición a la rabia humana / Equivalence between pre-exposure schemes for human rabies and evaluation of the need for serological monitoring

OBJETIVO: Avaliar a resposta imune humoral do esquema de pré-exposição da raiva humana realizado pelas vias intramuscular e intradérmica e a necessidade de sorologia de controle. MÉTODOS: Estudo de intervenção controlado e randomizado, realizado em São Paulo, SP, em 2004-2005. Foram recrutados 149 voluntários, dos quais 127 (65 intradérmica e 62 intramuscular) completaram o esquema de vacinação e realizaram avaliação da resposta imune humoral dez, 90 e 180 dias após o término da vacinação. Foram considerados dois desfechos para a comparação entre as duas vias de aplicação: a média geométrica do título de anticorpos neutralizantes e a proporção de indivíduos com títulos satisfatórios (> 0,5 UI/mL) em cada momento de avaliação. Foi analisada a associação da resposta humoral com dados antropométricos e demográficos por meio de teste de médias e qui-quadrado com correção de Yates. Após a conclusão do esquema foram feitas a comparação da proporção de soropositivos pelo teste de Kruskall Wallis e a comparação dos títulos médios por análise de variância. RESULTADOS: Os títulos médios de anticorpos foram maiores nos indivíduos que receberam as vacinas por via intramuscular. A percentagem de voluntários com títulos satisfatórios (> 0,5 UI/mL) diminuiu com o tempo em ambos os grupos, porém, no grupo que recebeu as vacinas por via intradérmica, a proporção de títulos satisfatórios no dia 180 variou de 20 por cento a 25 por cento, enquanto pela via intramuscular variou de 63 por cento a 65 por cento. Não se observou associação da resposta imune humoral com as variáveis demográficas ou antropométricas. CONCLUSÕES: A sorologia após a terceira dose pode ser considerada desnecessária em indivíduos sob controle quanto à exposição, uma vez que 97 por cento e 100 por cento dos voluntários vacinados, respectivamente por via intradérmica e pela via intramuscular, apresentaram níveis de anticorpos satisfatórios (> 0,5 UI/mL).

OBJECTIVE: To evaluate the humoral immune response to the pre-exposure schedule of human rabies vaccination through intradermal and intramuscular routes, as well as the need for serological monitoring. METHODS: A randomized and controlled intervention study was carried out in São Paulo, Southeastern Brazil, from 2004-2005. There were 149 volunteers, of which 127 completed the vaccination schedule (65 intradermal and 62 intramuscular) and underwent humoral immune response evaluation at ten, 90 and 180 days post-vaccination. Two outcomes were considered for comparing the two routes of administration: the geometric average of neutralizing antibody titers and the proportion of individuals with satisfactory titers (> 0.5 IU/mL) at each evaluation point. The association of the humoral immune response with anthropometric and demographic data was analyzed through a normal distribution test and a chi-square test with a Yates correction. After completion of the vaccination schedule, the proportion of seropositive results was compared by the Kruskall Wallis test, and the average titers were compared by variance analysis. RESULTS: the average antibody titers were higher in patients who were vaccinated intramuscularly. The percentage of volunteers with satisfactory titers (> 0.5 percent IU/mL) decreased over time in both groups. However, in the group vaccinated intradermally the rate of satisfactory titers on day 180 ranged from 20 percent to 25 percent, while the intramuscular route varied from 63 percent to 65 percent. An association between the humoral immune response and the demographic and anthropometric variables was not observed. CONCLUSIONS: Serology after the third dose can be considered unnecessary in unexposed patients, since 97 percent and 100 percent of volunteers respectively vaccinated by the intradermal and intramuscular route presented satisfactory antibody levels (> 0.5 percent IU/mL).

OBJETIVO: Evaluar la respuesta inmune humoral del esquema de pre-exposición de la rabia humana realizado por las vías intramuscular e intradérmica y la necesidad de serología de control. MÉTODOS: Estudio de intervención controlado y aleatorio, realizado en Sao Paulo, Sureste de Brasil, en 2004-2005. Fueron reclutados 149 voluntarios, de los cuales 127 (65 intradérmica y 62 intramuscular) completaron el esquema de vacunación y realizaron evaluación de la respuesta inmune humoral 10, 90 y 180 días posterior al término de la vacunación. Fueron considerados dos resultados para la comparación entre las dos vías de aplicación: el promedio geométrico del título de anticuerpos neutralizantes y la proporción de individuos con títulos satisfactorios (> 0,5 UI/mL) en cada momento de la evaluación. Fue analizada la asociación de la respuesta humoral con datos antropométricos y demográficos por medio de prueba de medias y chi-cuadrado con corrección de Yates. Posterior a la conclusión del esquema fueron realizadas la comparación de la proporción de seropositivos por la prueba de Kruskall Wallis y la comparación de los títulos promedios por análisis de varianza. RESULTADOS: Los títulos promedios de anticuerpos fueron mayores en los individuos que recibieron las vacunas por vía intramuscular. El porcentaje de voluntarios con títulos satisfactorios (> 0,5 UI/mL) disminuyó con el tiempo en ambos grupos, sin embargo, en el grupo que recibió vacuna por vía intradérmica la proporción de títulos satisfactorios en el día 180 varió de 20 por ciento a 25 por ciento, mientras que por la vía intramuscular varió de 63 por ciento a 65 por ciento. No se observó asociación de la respuesta inmune humoral con las variables demográficas o antropométricas. CONCLUSIONES: La serología posterior a la tercera dosis puede ser considerada innecesaria en individuos bajo control con respecto a la exposición, una vez que 97 por ciento y 100 por ciento de los voluntarios vacunados...

ELISA suitable for the detection of rabies virus antibodies in serum samples from human vaccinated with either cell-culture vaccine or suckling-mouse-brain vaccine

O metodo imunoenzimatico (ELISA) foi adaptado para quantificar anticorpos anti-rabicos em soros de pessoas previamente imunizadas. Foi utilizado como antigeno, particulas virais purificadas inativadas, e como conjugado, Proteina A conjugada a peroxidase. Foram testados soros de pessoas vacinadas com vacina de cultura celular ou com vacina produzida em cerebro de camundongo. Os resultados foram comparados a aqueles obtidos pela prova de soroneutralizacao em cultura celular. A media e o desvio padrao foram calculados para 126 soros negativos e para 73 soros de pessoas vacinadas mas com titulo menor que 0,5 UI/ml. Foi proposta a adocao de uma regiao de duvida, levando a uma diminuicao de resultados falso positivos. A sensibilidade, especificidade e concordancia do teste foram respectivamente: 87,5 por cento, 92,4 por cento e 88,5 por cento. Nao foram observadas diferencas significativas quando comparados os resultados dos individuos vacinados com uma ou outra vacina utilizada...

Studies on human anti-rabies immunization in Brazil: II-preliminary evaluation of the 2-1-1 schedule for human pre-exposure anti-rabies immunization, employing suckling mouse brain vaccine

Este estudo apresenta resultados preliminares de titulos de anticorpos neutralizantes (AcN) obtidos em diferentes dias durante imunizacao anti-rabica humana empregando o esquema 2-1-1 (uma dose administrada em cada deltoide no dia 0, e uma dose nos dias 7 e 21), recomendado pela OMS para tratamento pos-exposicao com vacinas de cultivo celular...