Evolução do diagnóstico virológico de raiva humana no Instituto Pasteur de São Paulo, Brasil (1970-2020): análise de dados / Evolution of the virological diagnosis of human rabies at the Pasteur Institute of São Paulo, Brazil (1970-2020): data analysis

O diagnóstico laboratorial precoce de raiva humana deve ser realizado por testes apropriados, visto que a aplicação de protocolos de tratamento médico em indivíduos internados depende dos resultados laboratoriais. O presente estudo analisou os dados referentes aos 560 casos suspeitos de raiva humana submetidos ao diagnóstico virológico no IP-SP entre os anos de 1970 e 2020. Houve um avanço das metodologias laboratoriais, especialmente as moleculares, que passaram a ser essenciais, possibilitando o tratamento de indivíduos expostos, bem como a determinação da fonte de infecção dos casos, fato fundamental para a efetividade de ações de controle em regiões vulneráveis à disseminação da doença. Intervenções no ciclo urbano da raiva, por meio de vacinação de cães e gatos e encaminhamento de amostras para diagnóstico, diminuiram os casos transmitidos por cães, principalmente no Sudeste. Em contrapartida, no mesmo período foi observado um aumento exponencial de casos relacionados ao ciclo silvestre nas regiões Norte (32%) e Nordeste (53,3%), tendo os morcegos como principais transmissores (72%), seguidos dos primatas não humanos (6%) e dos canídeos silvestres (1%). Esses resultados demonstraram a importância do aprimoramento do diagnóstico laboratorial, que é parte essencial na condução de estratégias de controle, bem como de tratamento de indivíduos expostos.

Early laboratory diagnosis of human rabies should be performed by appropriate tests, since the application of medical treatment protocols in hospitalized individuals depends on laboratory results. The present study analyzed the data referring to the 560 suspected cases of human rabies submitted to virological diagnosis in the IP-SP from 1970 to 2020. There has been an advance in laboratory methodologies, especially molecular ones, which have become essential, enabling the treatment of exposed individuals, as well as allowing the determination of the source of infection of cases, a fundamental fact for the effectiveness of control actions in regions vulnerable to the spread of the disease. Interventions in the urban cycle of rabies, through vaccination of dogs and cats and referral of samples for diagnosis decreased the cases transmitted by dogs, especially in the Southeast, on the other hand, an exponential increase of cases was observed in the same period, in the North (32%) and Northeast (53.3%) regions, with cases related to the wild cycle, with bats as the main transmitters (72%), followed by non-human primates (6%), and wild canids (1%). Our results demonstrated the importance of improving laboratory diagnosis, which is an essential part of conducting control strategies as well as the treatment of exposed individuals.

Monoclonal antibodies for characterization of rabies virus isolated from non-hematophagous bats in Brazil.

INTRODUCTION: In Brazil, various isolates of rabies virus (RABV) show antigenic profiles distinct from those established by the reduced panel of eight monoclonal antibodies (MAbs) determined by the Centers for Disease Control and Prevention (CDC), utilized for the antigenic characterization of RABV in the Americas. The objective of this study was to produce MAbs from RABV isolates from insectivorous bats with an antigenic profile incompatible with the pre-established one. METHODOLOGY: An isolate of RABV from the species Eptesicus furinalis that showed an antigenic profile incompatible with the panel utilized was selected. Hybridomas were produced utilizing the popliteal lymph nodes of mice immunized with ribonucleoproteins purified from the isolate. RESULTS: Two MAbs-producing clones were obtained, BR/IP1-3A7 and BR/IP2-4E10. Fifty-seven isolates of RABV from different species of animals and different regions of Brazil were analyzed utilizing the MAbs obtained. In the analysis of 23 RABV isolates from non-hematophagous bats, the MAbs cross-reacted with ten isolates, of which four were of the species Nyctinomops laticaudatus, one of the species Eptesicus furinalis, and five of the genus Artibeus. Of the nine isolates of non-hematophagous isolates that displayed an incompatible profile analyzed, characteristic of insectivorous bats, BR/IP1-3A7 reacted with five (55.55%) and BR/IP2-4E10 with four (44.44%). CONCLUSIONS: The MAbs obtained were able to recognize epitopes common between the three genera, Artibeus, Eptesicus, and Nyctinomops, thereby allowing the antigenic characterization of RABV isolates in Brazil.

Bufotenine is able to block rabies virus infection in BHK-21 cells.

BACKGROUND: Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella. METHODS: Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity, Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine. RESULTS: In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action. CONCLUSIONS: This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action.Conclusions This work do not present or propose bufotenine as a drug for the treatment of rabies due to the hallucinogen and psychotropic effects of the molecule. However, continued studies in the elucidation of the antiviral mechanism of this molecule, may lead to the choice or development of a tryptamine analogue presenting potential clinical use.(AU)

Bufotenine is able to block rabies virus infection in BHK-21 cells

Background Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, and kills more than 55.000 people every year. It is caused by an enveloped single stranded RNA virus that affects the central nervous system, through an infection initiated by the muscular nicotinic acetylcholine receptor, according to many authors. Alkaloids, such as acetylcholine, are widespread molecules in nature. They are present in numerous biological fluids, including the skin secretion of many amphibians, in which they act (together with proteins, peptides and steroids) as protection agents against predators and/or microorganisms. Among those amphibians that are rich in alkaloids, there is the genus Rhinella.Methods Bufotenine was isolated from Rhinela jimi skin secretion after a liquid-liquid partition (H2O:CH2Cl2) and reversed phase high-performance liquid chromatography analyses (RP-HPLC). Bufotenine was also extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC, as well. Structural characterization was performed by mass spectrometry and nuclear magnetic resonance analyses. Cytotoxic tests of bufotenine were performed over baby hamster kidney (BHK-21) cells using MTT test. For the antiviral activity,Rabies virus strain Pasteur vaccine (PV) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and bufotenine.Results In the present work we describe the effects of bufotenine, obtained either from toads or plants, that can inhibit the penetration of rabies virus in mammalian cells through an apparent competitive mechanism by the nicotinic acetylcholine receptor. Moreover, this inhibition was dose- and time-dependent, pointing out to a specific mechanism of action...

Infecção de células BHK-21 cultivadas em monocamadas estacionárias por cepas de vírus PV e CVS / Infection of BHK-21 cells cultivated in stationary monolayers by PV and CVS strains

O vírus da raiva pode estar presente em diferentes tecidos e órgãos, tornandopossível a sua replicação em diversos tipos de culturas celulares. Considerando a fundamental importância da produção desse vírus in vitro para a realização detestes diagnósticos, produção de soros hiperimunes e pesquisas, o objetivo deste estudo foi avaliar a infecção viral das cepas PV (Pasteur Virus) e CVS (Challenge Virus Standard) em linhagem de células BHK-21 (Baby Hamster Kidney). As células foraminfectadas com as cepas PV e CVS e cultivadas em frascos estacionários de cultivo celular e em microplacas com lamínulas, a 37ºC por até 96 horas. A cada três horas foram coletadas alíquotas do sobrenadante dos frascos, para acompanhamento daconcentração das partículas virais, e lamínulas para avaliar a infecção viral nas células. As avaliações foram realizadas por imunofluorescência direta, para definição da maior diluição em que as suspensões virais infectaram 100% da monocamada confluente de células BHK-21 e para avaliar o aumento da intensidade de fluorescência, expressa em cruzes (+ a ++++), identificando o antígeno viral, demonstrado por fotodocumentação. A presença de partículas viraisfoi observada a partir de nove horas pós-infecção, em ambas as cepas. As partículas virais das cepas PV e CVS no sobrenadante foram obtidas a partir de 15 e 18 horas de incubação, respectivamente, sendo observada a maior concentração de partículasnas suspensões virais das duas cepas, 72 horas pós-infecção. Portanto, o protocolo usado demonstrou eficiência, independente da cepa empregada, permitindo a obtenção de bons títulos nas suspensões virais produzidas

Diagnóstico ante-mortem da raiva humana: anticorpos neutralizantes em soro e líquido cefaloraquidiano / Diagnosis of human rabies: virus-neutralizing antibodies in serum and cerebrospinal fluid

O diagnóstico laboratorial ante-mortem da raiva humana envolve a pesquisa do vírus em folículo piloso, saliva, líquido cefalorraquidiano (LCR) e impressões de córnea, e a pesquisa de anticorpos neutralizantes do vírus da raiva (AcN) em amostras de soro e LCR. A presença de AcN no soro ou LCR de indivíduos não vacinados é indicativa de raiva, porém, esses resultados só ocorrem nos estágios finais da doença. Neste estudo foram analisados os resultados da pesquisa de AcN em amostras de soro e/ou LCR de três pacientes com suspeita de raiva, sem histórico de sorovacinação. O método de pesquisa de AcN foi o microteste simplificado de inibição de fluorescência. O paciente A, apesar de não ter tido análise de sistema nervoso central (SNC), teve diagnóstico de raiva com base nos sintomas clínicos e títulos de AcN de 3,0 UI/mL no soro e 0,37 UI/mL no LCR. Dos dois pacientes que tiveram o vírus identificado post-mortem no SNC, o paciente B apresentou LCR com título de 12,0 UI/mL de AcN e o paciente C apresentou resultados negativos de AcN no soro e no LCR, sendo compatíveis com a relação existente entre coleta e período de morbidade. Esses resultados mostram que a pesquisa de AcN de pacientes suspeitos de raiva, sem histórico de sorovacinação e com longo período de morbidade, deve ser feita em coletas subseqüentes de soro e LCR, para possibilitar o diagnóstico ante-mortem da raiva, especialmente quando a coleta post-mortem de SNC tornar-se inviável.

Purificação de vírus da raiva e ribonucleoproteínas para produção de conjugados fluorescentes / Rabies virus and ribonucleoprotein purification for fluorescent conjugate production

A Organização Mundial da Saúde recomenda o teste de imunofluorescência direta (IFD) para a pesquisa do vírus da raiva em sistema nervoso central (SNC) de animais suspeitos e o teste rápido de inibição de focos fluorescentes (RFFIT) para a avaliação do título de anticorpos neutralizantes, o qual foi adaptado no Brasil para o microteste simplificado de inibição de fluorescência (SFIMT). Esses testes podem ser revelados com o uso de conjugados fluorescentes anti-vírus da raiva (CA-VR) ou anti-ribonucleoproteínas (CA-RNPs). A obtenção de conjugado fluorescentes é feita por meio de conjugação do fluorocromo isotiocianato de fluoresceína (ITCF) a anticorpos anti-vírus da raiva ou anti-RNPs purificados. Os métodos utilizados para purificação de vírus da raiva e ribonucleoproteínas foram ultracentrifugação em gradiente de sacarose e gradiente de Cloreto de Césio, respectivamente. Para obtenção de anticorpos anti-vírus da raiva e anti-RNPs dois grupos de coelhos foram imunizados e um de cada grupo foi selecionado de acordo com os maiores títulos, 3600 UI/mL pelo teste de SFIMT e diluição de 1/2560 pelo teste de imunofluorescêscia indireta. Os anticorpos foram purificados por cromatografia de troca iônica (QAE-Sephadex A 50), conjugados ao ITCF e filtrados por cromatografia de peso molecular (G-50 Sephadex). As titulações dos lotes dos conjugados, realizadas por IFD e SFIMT, apresentaram títulos de 1/200 e 1/250 com o lote antiVR e 1/400 e 1/500 com o lote anti-RNPs. Testes de sensibilidade e especificidade dos conjugados produzidos foram realizados por IFD em 100 amostras de tecido de SNC de diferentes espécies animais, apresentando resultados 100% concordantes entre si e com os resultados do conjugado comercial utilizado como padrão. A qualidade dos conjugados avaliada por fotografias demonstrou fluorescência intensa e específica e ausência de fluorescência nos controles negativos.

Antibody response in cattle after vaccination with inactivated and attenuated rabies vaccines

Despite the absence of current official reports showing the number of cattle infected by rabies, it is estimated that nearly 30,000 bovines are lost each year in Brazil. In order to minimize the important economic losses, control of the disease is achieved by eliminating bat colonies and by herd vaccination. In this study, we compare the antibody response in cattle elicited by vaccination with an attenuated ERA vaccine (AEvac) and an inactivated-adjuvanted PV (IPVvac) vaccine. The antibody titers were appraised by cell-culture neutralization test and ELISA, and the percentage of seropositivity was ascertained for a period of 180 days. IPVvac elicited complete seropositivity rates from day 30 to day 150, and even on day 180, 87 per cent of the sera showed virus-neutralizing antibody titers (VNA) higher than 0.5IU/ml. There were no significant differences between the VNA titers and seropositivity rates obtained with IPVvac in the two methods tested. AEvac, however, elicited significantly lower titers than those observed in the group receiving inactivated vaccine. In addition, the profiles of antirabies IgG antibodies, evaluated by ELISA, and VNA, appraised by cell-culture neutralization test, were slightly different, when both vaccines were compared.