Publications by authors named "Andrea de Cássia Rodrigues da Silva"

9 Publications

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Performance evaluation of the polyclonal anti-rabies virus ribonucleoprotein IgG antibodies produced in-house for use in direct fluorescent antibody test.

J Virol Methods 2020 06 1;280:113879. Epub 2020 May 1.

Instituto Pasteur, São Paulo, Brazil. Electronic address:

Fluorescein isothiocyanate (FITC) labelled anti-rabies virus ribonucleoprotein (RNP) antibodies can be used as immunoreagents in direct fluorescent antibody testing (dFAT) for rabies diagnoses. While in-house products are occasionally used by laboratories, most conjugates are commercial reagents. Commercial anti-RNP antibodies are only available for research purposes in Brazil, however, which contributes to the increasing use of in-house produced antibodies. Considering that conjugate quality may influence the results obtained during rabies diagnosis, we sought to analyze the performance requirements of in-house produced polyclonal anti-RNP IgG-FITC for application in dFAT. To that end, their reproducibility, diagnostic sensitivity, and specificity were evaluated. The titer of polyclonal anti-RNP IgG-FITC was initially determined and evaluated by dFAT, using central nervous system (CNS) samples of different animal species (dogs, cats, bovines, equines, bats, and non-human primates). As our main result, the polyclonal anti-RNP IgG-FITC reached a titer of 1:30/1:40 in dFAT, with 100% of diagnostic sensitivity and specificity. In terms of reproducibility, the antibodies, regardless the production batch, presented the same performances. In conclusion, the in-house produced polyclonal anti-RNP IgG-FITC proved suitable for rabies virus antigen detection by dFAT.
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http://dx.doi.org/10.1016/j.jviromet.2020.113879DOI Listing
June 2020

Evaluation of polyclonal anti-RNP IgG antibody for rabies diagnosis by indirect rapid immunohistochemistry test.

Acta Trop 2020 Jun 21;206:105340. Epub 2020 Feb 21.

Instituto Pasteur, 393, Paulista Avenue, 01311-000, São Paulo, Brazil. Electronic address:

Rabies still represents a major public health threat and estimated to cause 60,000 human deaths annually, particularly in developing countries. Thus, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. The WHO and OIE recommended gold standard diagnostic technique for rabies is the direct immunofluorescence assay (dFAT). However, dFAT is expensive and requires a high level of expertise. As an alternative, the rapid immunohistochemistry technique is a promise to be a simple and cost effective diagnostic tool for rabies, and can be performed on field conditions prevalent in developing countries. However, no validated commercial conjugate antibody for rabies is available to meet the laboratory demand. Here, we evaluated the polyclonal anti-rabies virus ribonucleoprotein (RNP) IgG antibody for Rabies lyssavirus (RABV) detection by indirect rapid immunohistochemistry test (iRIT). We tested polyclonal anti-RNP IgG antibody against a batch of 100 brain specimens representing a wide phylogenetic origin in the State of São Paulo, Brazil. The purified IgG obtained 100% of diagnostic specificity and sensibility for RABV antigen detection in iRIT compared with the gold standard dFAT. In conclusion, our results demonstrate that the polyclonal anti-RNP IgG antibody may be used as a diagnostic reagent for rabies using iRIT, with the expectation of increase in availability and cost reduction of the epidemiological surveillance for developing countries.
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http://dx.doi.org/10.1016/j.actatropica.2020.105340DOI Listing
June 2020

Rabies surveillance in wild mammals in South of Brazil.

Transbound Emerg Dis 2020 Mar 21;67(2):906-913. Epub 2019 Nov 21.

Departamento de Microbiologia, Imunologia e Parasitologia, Instituto de Ciências Básicas da Saúde (ICBS) Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil.

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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http://dx.doi.org/10.1111/tbed.13415DOI Listing
March 2020

Purification of IgG against ribonucleoprotein by a homemade immunoaffinity chromatography column for rabies diagnosis.

J Immunol Methods 2019 08 20;471:1-10. Epub 2019 Mar 20.

Instituto Pasteur, São Paulo, Brazil. Electronic address:

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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http://dx.doi.org/10.1016/j.jim.2019.03.007DOI Listing
August 2019

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

J Venom Anim Toxins Incl Trop Dis 2015 2;21:50. Epub 2015 Dec 2.

Butantan Institute, Laboratory of Biochemistry and Biophysics, Av. Vital Brazil, 1500, São Paulo, SP 05503-900 Brazil.

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.
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http://dx.doi.org/10.1186/s40409-015-0048-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668702PMC
December 2015

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

J Infect Dev Ctries 2015 Nov 30;9(11):1238-49. Epub 2015 Nov 30.

Instituto Pasteur, São Paulo, SP, Brasil.

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.
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http://dx.doi.org/10.3855/jidc.6959DOI Listing
November 2015

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

J Venom Anim Toxins Incl Trop Dis 2014 13;20(1):45. Epub 2014 Oct 13.

Laboratory of Biochemistry and Biophysics, Butantan Institute, São Paulo, SP Brazil.

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.
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http://dx.doi.org/10.1186/1678-9199-20-45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203886PMC
October 2014

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

Rev Saude Publica 2010 Jun 7;44(3):548-54. Epub 2010 May 7.

Instituto Pasteur, São Paulo, SP, Brazil.

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).
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http://dx.doi.org/10.1590/s0034-89102010005000005DOI Listing
June 2010

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

J Clin Lab Anal 2009 ;23(1):7-13

Rabies Diagnostic Laboratory, Instituto Pasteur de São Paulo, São Paulo, 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.
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http://dx.doi.org/10.1002/jcla.20275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6649073PMC
August 2009