Seroepidemiological survey to cell culture rabies vaccines (CCRV) in Brazil.

Rabies is a contagious viral disease that can be easily transmitted by the saliva and brain/nervous system tissues of the infected animals, causing severe and fatal encephalitis in both animals and humans. Vaccination campaigns are crucial to combat and prevent rabies's spread in dogs and humans. The Modified Fuenzalida & Palicios vaccines have been widely used since the 70s and have proven effective in producing a solid serological response. Since 2008, the Brazilian Ministry of Health has introduced a Cell Culture Rabies Vaccine (CCRV) for all dog mass vaccination campaigns in Brazil. However, to date, there is limited evidence on the immunologic response of dogs to this type of vaccine in field conditions. The present study evaluated the serological response in dogs vaccinated with CCRV from blood samples of 724 dogs using the Simplified Fluorescence Inhibition Microtest - SFIMT. Dogs with a titer equal to 0.5 IU/mL or above were considered seropositive. The results revealed that 59.12% (428/724) of all dogs tested and 48.49% (32/66) of primo-vaccinated animals were seropositive. The percentage of seronegative animals was higher than seropositive for animals that received a single dose during their life (p < 0.05). The opposite was observed in animals with five or more doses. The results of this study demonstrated that the CCRV vaccines elicit a satisfactory immunological response in field conditions and can constitute an essential population-level preventive strategy as part of annual canine rabies vaccination campaigns. Although its effectiveness has been studied, there is limited evidence of its immunological response in dogs under field conditions. This paper evaluates the serological response to CCRV in dogs vaccinated during mass vaccination campaigns from 2012 to 2017.

EXPOSURE OF WILD BOAR (SUS SCROFA) TO THE COMMON VAMPIRE BAT AND LACK OF IMMUNE PROTECTION TO RABIES VIRUS IN BRAZILIAN HUNTERS.

Rabies virus is recognized as one of the most fatal zoonotic agents affecting all mammals. Wild boars (Sus scrofa), classified as a large-size exotic invasive species in Brazil with nationwide hunting permitted, may serve as an extra blood source for the common vampire bat (Desmodus rotundus). Our aim was to document wild boar exposure to vampire bats to determine the seroprevalence of rabies virus antibodies in wild boars and to determine the immune status of hunters in southern and central-western Brazilian regions. Serum samples were collected from 80 wild boars and 49 hunters from natural and degraded areas of the Atlantic Forest biome of southern Brazil and in degraded areas of the Cerrado biome of central-western Brazil. The rabies-modified rapid fluorescent focus inhibition test was performed to detect the presence of rabies virus neutralizing antibodies in wild boars and considered seropositive when ≥0.10 IU/mL. The simplified fluorescence inhibition microtest was used for samples from hunters with a titer of ≥0.50 IU/mL and considered indicative of seroconversion. While 11% (9/80) of wild boars had serum titers for rabies exposure (≥0.10 IU/mL), 88% (43/49) of corresponding hunters lacked immune protective titers (<0.50 IU/mL). Wild boars showed serum titers for rabies likely due to contact with contaminated saliva of vampire bats or from infected carcass consumption. Additionally, Brazilian wild boars can be exposed to rabies and may play an important role in the sylvatic rabies cycle by providing a blood supply for vampire bats, highlighting the possibility of direct transmission of rabies virus to hunting dogs and hunters. These findings suggested hunters are a potential risk group for contracting rabies, and the World Health Organization may consider adding this occupation to their recommendations of who should receive the pre-exposure rabies vaccination.

Rabies Virus Exposure in Wild Lowland Tapirs (Tapirus terrestris) from Three Brazilian Biomes.

We evaluated the presence of antibodies for rabies virus in 177 serum samples from 125 wild lowland tapirs (Tapirus terrestris) from three different Brazilian biomes. The rapid fluorescent focus inhibition test was performed. No antibody titers suggesting the circulation of the rabies virus in tapir habitat were detected.

Development of biotinylated polyclonal anti-ribonucleoprotein IgG for detection of rabies virus antigen by direct rapid immunohistochemical test.

The direct rapid immunohistochemical test (dRIT) has been recommended for laboratorial diagnosis of rabies, especially in developing countries. The absence of commercial primary antibodies, however, still represents a major limitation to its wider use in testing. We describe here the development of a biotinylated polyclonal antibody against Rabies lyssavirus (RABV) ribonucleoprotein (RNP) and its use as a primary reagent in dRIT. Anti-RNP polyclonal horse IgG was purified by ionic exchange chromatography followed by immunoaffinity column chromatography, and its affinity, diagnostic sensitivity, and specificity were evaluated. CNS samples (120) of suspected rabies cases in different animal species were tested by dRIT, with the positive (n = 14) and negative (n = 106) results confirmed by direct fluorescence antibody testing (dFAT). Comparing the results of dRIT and dFAT, we found that the biotinylated anti-RNP IgG delivered 100% diagnostic specificity and sensibility for rabies diagnosis. Our findings show that the biotinylated anti-RNP polyclonal IgG can be produced with the quality required for application in dRIT. This work represents an important step in efforts to diagnose rabies in developing countries.

Large protein as a potential target for use in rabies diagnostics

Rabies is a zoonotic viral disease that remains a serious threat to public health worldwide. The rabies lyssavirus (RABV) genome encodes five structural proteins, multifunctional and significant for pathogenicity. The large protein (L) presents well-conserved genomic regions, which may be a good alternative to generate informative datasets for development of new methods for rabies diagnosis. This paper describes the development of a technique for the identification of L protein in several RABV strains from different hosts, demonstrating that MS-based proteomics is a potential method for antigen identification and a good alternative for rabies diagnosis.(AU) i

Delayed progression of rabies transmitted by a vampire bat.

Here, we compared the growth kinetics, cell-to-cell spread, and virus internalization kinetics in N2a cells of RABV variants isolated from vampire bats (V-3), domestic dogs (V-2) and marmosets (V-M) as well as the clinical symptoms and mortality caused by these variants. The replication rate of V-3 was significantly higher than those of V-2 and V-M. However, the uptake and spread of these RABV variants into N2a cells were inversely proportional. Nevertheless, V-3 had longer incubation and evolution periods. Our results provide evidence that the clinical manifestations of infection with bat RABV variant occur at a later time when compared to what was observed with canine and marmoset rabies virus variants.

Rabies lyssavirus Isolates from Brazilian Different Reservoirs Species Present Distinct Pattern of Propagation in N2a Cell

Background: Rabies cell culture infection test was developed for the isolation of Rabies lyssavirus and as an alternative for the mouse inoculation test. However, tissue culture for street rabies strains produces low viral titer. Here, we assessed the quantity of brain tissue for successful viral isolation toward increased virus titer in effective way.Methods: Brain tissue isolates from different reservoirs species of Brazil were harvested in different concentration and inoculated in mouse neuroblastoma cells (N2a). These isolates were measured infectious viral titer and cell viability followed by consecutive passages in N2a cells.Results: Inoculum containing were prominent Rabies lyssavirus due to higher viral titer and not significantly dead cell. After consecutive passages in N2a cells Rabies lyssavirus variant maintained by vampire bat had remarkable adaptation to the culture system, while isolates from marmoset presents distinct pattern of propagation in N2a cell when compared with other groups.Conclusion: Based on these results, the isolation followed by viral replication assay may be used in isolates from different reservoirs which enable an effective amplification of the wild type virus strains

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 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.

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.

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)

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.

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...

Equivalence between pre-exposure schemes for human rabies and evaluation of the need for serological monitoring.

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% 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% to 25%, while the intramuscular route varied from 63% to 65%. 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% and 100% of volunteers respectively vaccinated by the intradermal and intramuscular route presented satisfactory antibody levels (> 0.5% IU/mL).

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

First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil.

The laboratory tests recommended by the World Health Organization for detection of rabies virus and evaluation of specific antibodies are performed with fluorescent antibodies against the virus, the ribonucleoproteins (RNPs), or by monoclonal antibodies. In this study, we purified the rabies virus RNPs for the production of a conjugate presenting sensibility and specificity compatible with commercial reagents. The method employed for the purification of RNPs was ultracentrifugation in cesium chloride gradient, the obtained product being used for immunizing rabbits, from which the hyperimmune sera were collected. The serum used for conjugate production was the one presenting the highest titer (1/2,560) when tested by indirect immunofluorescence. The antibodies were purified by anion exchange chromatography (QAE-Sephadex A-50),conjugated to fluorescein isothiocyanate and separated by gel filtration (Sephadex G-50). The resulting conjugate presented titers of 1/400 and 1/500 when assayed by direct immunofluorescence (DIF) and simplified fluorescence inhibition microtest, respectively. Sensibility and specificity tests were performed by DIF in 100 central nervous system samples of different animal species, presenting 100% matches when compared with the commercial reagent used as standard, independent of the conservation state of the samples. The quality reached by our conjugate will enable the standardization of this reagent for use by the laboratories performing diagnosis of rabies in Brazil, contributing to the intensification of the epidemiological vigilance and research on this disease.

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.

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...

Resposta imune humoral na imunização anti-rábica humana: comparação de títulos de anticorpos neutralizantes de isotipos de imunoglobulinas e de avidez de IgC na vacinação intramuscular e intradermica / Immune response

Os esquemas de tratamento anti-rábico humano utilizados no Brasil têm se caracterizado pela utilização de grande número de doses de vacina preparada em tecido cerebral de animais, tipo Fuenzalida & Palacios (F&P), administrados em dias consecutivos. Desde de 1992 a OMS desaconselha totalmente o uso desse tipo de vacina recomendando exclusivamente a utilização de vacinas de cultivo celular. Com o objetivo de fazer uma ampla caracterização da resposta imune humoral decorrente da vacinação anti-rábica em humanos, para fornecer subsídios para futuras alterações nas normas de tratamento humano estudamos os esquemas preconizados pela Secretaria de Estado da Saúde de São Paulo e Fundação Nacional de Saúde incluindo os tratamentos pós-exposição (“10+3” tratamento que inclui aplicação de soro e “7+2” tratamento sem aplicação de soro) e o esquema pré-exposição (“3+1”). A resposta produzida com os tratamentos utilizados rotineiramente no Brasil foi comparada com as respostas ao tratamento pré-exposição (0-7-28) com vacina de cultivo celular (HDCV), administrado por via intramuscular (IM) ou intradérmica (ID), conforme preconizado pela OMS. Foram analisadas um total de 543 amostras de soro humano, selecionados da soroteca do Setor de Sorologia do Instituto Pasteur de São Paulo. Estes soros foram coletados de indivíduos vacinados contra a raiva com diferentes esquemas de imunização. Os títulos de anticorpos neutralizantes (AcN) foram obtidos através do microteste simplificado de inibição de focos fluorescentes e as dosagens dos tipos e subtipos de imunoglobulinas (Ig) foram obtidas pelo método de ELISA. Os títulos de AcN ao longo do tempo foram semelhantes para os dois esquemas IM e ID com HDCV não ocorrendo diferenças significantes. No dia 7, com o esquema IM, obtivemos 5,6% de soroconversão e com o ID, 2,6%. Ambos esquemas mostraram 100% de soroconversão no dia 28. Os títulos de AcN obtidos aproximadamente 10 dias após a administração dos três esquemas com vacina do tipo F&P mostraram-se semelhantes. Apenas o esquema 10+3 apresentou soroconversão menor que 100%. O reforço intradérmico com HDCV nos indivíduos que tinham recebido 1 ano antes um tratamento pré-exposição com vacina F&P, resultou em 100% de soroconversão (média geométrica = 23,78UI/ml). Para o esquema com HDCV a produção de anticorpos do isotipo IgG nos dias 7 e 28 foi semelhante para ambas as vias IM e ID

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...