RESUMO

Bufotenine, an alkaloid that can be found in plant extracts and skin secretions of amphibians, is reported to have potential antiviral activity. The present study evaluated the antiviral activity of bufotenine against different genetic lineages of rabies virus (RABV, a single-stranded, negative-sense RNA virus), canine coronavirus (CCoV, a positive-sense RNA virus) and two double-stranded DNA viruses (two strains of herpes simplex virus type 1/HSV-1 [KOS and the acyclovir-resistant HSV-1 strain 29R] and canine adenovirus 2, CAV-2). The maximal non-toxic bufotenine concentrations in Vero and BHK-21 cells were determined by MTT assays. The antiviral activity of bufotenine against each virus was assessed by examination of reductions in infectious virus titres and plaque assays. All experiments were performed with and without bufotenine, and the results were compared. Bufotenine demonstrated significant RABV inhibitory activity. No antiviral action was observed against CCoV, CAV-2 or HSV-1. These findings indicate that the antiviral activity of bufotenine is somewhat linked to the particular infectious dose used and the genetic lineage of the virus, although the mechanisms of its effects remain undetermined.

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The sylvatic cycle of rabies, caused by the Rabies lyssavirus (RABV), is maintained in the American Continent by aerial and terrestrial wild mammals. In this study, we combined passive surveillance of rescued wild animals with active serological surveillance in targeting areas at Rio Grande do Sul State and Santa Catarina State, south of Brazil, where bites of humans by wild animals have been reported. Circulation of RABV in Brazilian bats has been extensively demonstrated; however, the observation of such infections in unvaccinated terrestrial mammals is restricted to some regions of the Brazilian territory. The occurrence of rabies infection in unvaccinated animals has been identified by the detection of RABV antigens in brain tissues of dead animals or anti-rabies antibodies in live animals. Such strategies allow the surveillance of rabies and the assessment of spillover risks from infected animals to humans. Our aim included the identification of species of wild mammals that are involved in the sylvatic cycle of rabies virus in Southern Brazil and to assess the risk of rabies infection in patients bitten by wild animals in the state. To assess the anti-rabies seropositivity, sera were submitted to the Rapid Fluorescent Focus Inhibition Test (RFFIT). Among the 100 mammals tested, five animals were seropositive (5%) including three (one primate and two wild canids) with rabies virus neutralizing antibodies titres >0.5 IU/ml. Our results highlight the exposure to RABV of both primates and wild canids in Southern Brazil and suggest the occurrence of RABV exposure without the development of further symptoms. Further research should clarify the dynamics of rabies in wild canids and whether primates are accidental hosts or reservoirs for RABV at this region.

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RESUMO

Polyclonal or monoclonal antibodies against rabies virus ribonucleoprotein (RNP) conjugated to fluorescein isothiocyanate (FITC) have been employed for Rabies virus (RABV) antigen detection by the direct fluorescent antibody test (DFA). To date, these biomolecules have been purified by traditional methods such as precipitation by ammonium sulfate or ion exchange chromatography followed by ammonium sulfate precipitation, which allows only for partial detection of the protein of interest. In this study, we aimed to purify anti-RNP polyclonal horse IgG antibodies by cation-exchange chromatography in combination with a homemade immunoaffinity chromatography on RNP immobilized (RNP-IAC). Furthermore, to evaluate the accuracy of the prepared anti-RNP IgG fluorescent antibody in diagnostic purposes, DFA was applied for RABV antigen detection in suspected brain samples of different animal species. The combination of these two techniques made it possible to obtain antibodies with high selectivity and purity. Compared with the performance of the traditional method, anti-RNP IgG antibodies purified by RNP-IAC can be obtained from a smaller volume of hyperimmune serum and with greater avidity. Furthermore, the results obtained by DFA analyses revealed that the prepared anti-RNP IgG fluorescent antibody achieved 100% diagnostic specificity and sensitivity for RABV antigen detection. Thus, two-technique chromatographic, including RNP-IAC technology could be appropriate methods for the purification of polyclonal anti-RNP IgG for the use as a diagnostic reagent for rabies.

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O Programa Nacional de Profilaxia da Raiva tem como objetivo manter níveis imunogênicos protetores nos animais vacinados, com títulos de anticorpos neutralizantes (AcN) 0,5UI/mL. O objetivo deste estudo foi avaliar, de acordo com a idade, raça e o período entre a aplicação da vacina e a colheita do sangue, a resposta imunológica de cães que receberam dose única de vacina antivírus da raiva de cultivo celular. Neste estudo foram analisadas 432 amostras recebidas no Instituto Pasteur de São Paulo no triênio 2009-2011. Os dados foram analisados e a avaliação de AcN para o vírus da raiva foi realizada por meio do teste rápido de inibição de focos fluorescentes (RFFIT). Do total das amostras analisadas, 21,76% não possuíam títulos protetores. Dentre essas, 67,02% das amostras eram de filhotes e quando considerado o intervalo entre a data de aplicação da vacina e a colheita do sangue, 60,63% amostras não atingiram a titulação nos seis primeiros meses. Concluiu-se a partir do estudo desta amostragem a necessidade de uma segunda dose de vacina em filhotes, pois estes ficam mais suscetíveis à infecção pelo vírus da raiva, o que aumentaria a possibilidade de uma resposta rápida e duradoura.(AU)

The National Programme for Rabies Prevention aims to maintainimmunogenic protective levels in vaccinated animals, with titersof neutralizing antibodies (VNA) 0.5 IU / mL. The aim of thisstudy was to evaluate, according to age, race, and the periodof vaccination and blood collection, the immune response indogs that received a single dose of rabies vaccine virus in cellculture. In this study 432 samples received at the Pasteur Instituteof São Paulo in the triennium 2009-2011 were analyzed. Datawere analyzed and the evaluation of the VNA to rabies virus wasperformed by rapid fluorescent inhibition test foci (RFFIT). Of thetotal samples analyzed, 21.76% did not have protective titers.Among these, 67.02% samples were considered puppies andwhen the interval between the date of vaccine administration andblood collection, 60.63% samples did not reach the titration inthe first six months. It was concluded from this study sample theneed for a second dose of vaccine in puppies, as they are moresusceptible to infection by rabies virus, which would increase thepossibility of a rapid and durable response.(AU)

Assuntos

RESUMO

O Programa Nacional de Profilaxia da Raiva tem como objetivo manter níveis imunogênicos protetores nos animais vacinados, com títulos de anticorpos neutralizantes (AcN) 0,5UI/mL. O objetivo deste estudo foi avaliar, de acordo com a idade, raça e o período entre a aplicação da vacina e a colheita do sangue, a resposta imunológica de cães que receberam dose única de vacina antivírus da raiva de cultivo celular. Neste estudo foram analisadas 432 amostras recebidas no Instituto Pasteur de São Paulo no triênio 2009-2011. Os dados foram analisados e a avaliação de AcN para o vírus da raiva foi realizada por meio do teste rápido de inibição de focos fluorescentes (RFFIT). Do total das amostras analisadas, 21,76% não possuíam títulos protetores. Dentre essas, 67,02% das amostras eram de filhotes e quando considerado o intervalo entre a data de aplicação da vacina e a colheita do sangue, 60,63% amostras não atingiram a titulação nos seis primeiros meses. Concluiu-se a partir do estudo desta amostragem a necessidade de uma segunda dose de vacina em filhotes, pois estes ficam mais suscetíveis à infecção pelo vírus da raiva, o que aumentaria a possibilidade de uma resposta rápida e duradoura.

The National Programme for Rabies Prevention aims to maintainimmunogenic protective levels in vaccinated animals, with titersof neutralizing antibodies (VNA) 0.5 IU / mL. The aim of thisstudy was to evaluate, according to age, race, and the periodof vaccination and blood collection, the immune response indogs that received a single dose of rabies vaccine virus in cellculture. In this study 432 samples received at the Pasteur Instituteof São Paulo in the triennium 2009-2011 were analyzed. Datawere analyzed and the evaluation of the VNA to rabies virus wasperformed by rapid fluorescent inhibition test foci (RFFIT). Of thetotal samples analyzed, 21.76% did not have protective titers.Among these, 67.02% samples were considered puppies andwhen the interval between the date of vaccine administration andblood collection, 60.63% samples did not reach the titration inthe first six months. It was concluded from this study sample theneed for a second dose of vaccine in puppies, as they are moresusceptible to infection by rabies virus, which would increase thepossibility of a rapid and durable response.

Assuntos