Papers by Luciana B Chaves
Journal of Venomous Animals and Toxins including Tropical Diseases, 2014
Background: Rabies is a fatal zoonotic neglected disease that occurs in more than 150 countries, ... more 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 (H 2 O:CH 2 Cl 2) 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.
The proximity to domestic animals has been considered an important cause of disease of wildlife, ... more The proximity to domestic animals has been considered an important cause of disease of wildlife, and has led to recent epidemics in endangered species around the world. In this study, exposure to rabies virus in eleven freeliving jaguars (Panthera onca) captured from July 2010 to November 2012 in two protected areas in the Pantanal/MT/Brazil, was screened by Simplified Fluorescent Inhibition Microtest (SFIMT) and Rapid Fluorescent Focus Inhibition Test (RFFIT). Serum sample from each jaguar was analyzed twice in different days. Considering the presence of virus neutralizing antibodies (VNA) in samples with titers = 0,10, three jaguars had low positive titers for each test performed, for a frequency of 27.3%, but only a jaguar showed rabies‐ neutralizing antibodies on both SFIMT and RFFIT (0,19/0,12 and 0,14, respectively). Low titers of VNA have been detected in other species of wild carnivores, including apparently healthy free‐living jaguars, suggesting a nonlethal infection. In o...
Journal of Biological Chemistry, Mar 18, 1994
trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite t... more trans-Sialidase isolated from trypomastigote forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is multimeric and heterogeneous in size. We show here that limited proteolysis of tans-sialidase with papain yields a single monomeric polypeptide chain of 70 kDa that conserves full enzymatic activity on soluble and membrane-bound substrates. The papain fragment lacks most of the 12-amino acid repeats of the carboxyl-terminal domain that comprises about 50% of the native trans-sialidase. When injected into rabbits, the papain-generated fragment induces antibodies that inhibit trans-sialidase activity and trypomastigote sialylation. The repeats are also not required for the stability of the enzyme or for the correct folding during the biosynthesis in Escherichia coli, but seem essential for trans-sialidase oligomerization. We conclude that trans-sialidase is composed of two structurally and functionally independent domains.
Journal of virological methods, 2016
The most effective and sustainable method to control and eliminate rabies in wildlife is the oral... more The most effective and sustainable method to control and eliminate rabies in wildlife is the oral rabies vaccination (ORV) of target species, namely foxes and raccoon dogs in Europe. According to WHO and OIE, the effectiveness of oral vaccination campaigns should be regularly assessed via disease surveillance and ORV antibody monitoring. Rabies antibodies are generally screened for in field animal cadavers, whose body fluids are often of poor quality. Therefore, the use of alternative methods such as the enzyme-linked immunosorbent assay (ELISA) has been proposed to improve reliability of serological results obtained on wildlife samples. We undertook an international collaborative study to determine if the commercial BioPro ELISA Rabies Ab kit is a reliable and reproducible tool for rabies serological testing. Our results reveal that the overall specificity evaluated on naive samples reached 96.7%, and the coefficients of concordance obtained for fox and raccoon dog samples were 97....
Acta virologica
Rabies is a zoonotic viral disease that remains a serious threat to public health worldwide. The ... more 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.
This study investigates the exposure of free-living jaguars from two federal protected areas in t... more 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.
Page 1. Luciana Botelho Chaves Produção de anticorpos monoclonais para caracterização de variante... more Page 1. Luciana Botelho Chaves Produção de anticorpos monoclonais para caracterização de variantes antigênicas brasileiras de vírus da raiva Tese apresentada ao Programa de Pós-Graduação Interunidades em Biotecnologia ...
Anticorpos monoclonais (AcMo) contra proteínas do vírus da raiva (RABV) foram produzidos para ade... more Anticorpos monoclonais (AcMo) contra proteínas do vírus da raiva (RABV) foram produzidos para adequar a caracterização antigênica dos isolados no Brasil. Foram selecionados dois isolados de morcegos insetívoros, sendo um de Nyctinomops laticaudatus e outro de Eptesicus furinalis que apresentaram perfis não compatíveis (NC) com os pré-estabelecidos. As suspensões virais foram adaptadas para crescimento em cultura de células N2A. Para o preparo de AcMo foram utilizadas como antígeno as ribonucleoproteínas dos isolados selecionados. Foram obtidos dois AcMo, o 3A7 e o 4E10. Analisando 57 isolados de RABV com esses AcMo, o 3A7 reagiu com 21 (36,84%) e o 4E10 com 25 (43,85%). Dos 13 isolados caracterizados como variante antigênica 3 (Desmodus rotundus) o 3A7 reagiu com 8 (61,53%) e o 4E10 com 11 (84,61%). Dos 9 isolados com perfil NC em morcegos o 3A7 reagiu com 5 (55,55%) e o 4E10 com 4 (44,44%). Os anticorpos produzidos poderão auxiliar na complementação do painel existente de caracteri...
Molecular and Biochemical Parasitology, 1993
We have studied the trans-sialidase from insect forms of Trypanosoma cruzi growing in axenic cult... more We have studied the trans-sialidase from insect forms of Trypanosoma cruzi growing in axenic culture. Log phase epimastigotes expressed little or no trans-sialidase activity, and were unable to incorporate exogenous sialic acid. Transsalidase started to be expressed at the late logarithmic phase, with specific activity increasing steadily as the culture reached the stationary phase. Trans-sialidase was purified from the late log phase epimastigote culture, which contained less than 2% of metacyclic forms, yielding a glycoprotein that migrated as a single 90-kDa band in sodium dodecyl sulfate gels. This enzyme features: (1) no reaction with antibodies against the peptide repeats present in the carboxy-terminal of trypomastigote trans-sialidase; (2) positive reaction with antibodies raised against a fragment of trypomastigote trans-sialidase that contains the active site; (3) similar kinetic properties and identical acceptor-donor specificity when compared to the trypomastigote enzyme; and (4) neuraminidase activity in the absence of acceptors. Upon differentiation into metacyclic forms, a trans-sialidase activity containing the carboxy-terminal repeats of the trypomastigote enzyme was released into the medium. These results suggest that epimastigotes express a developmentally regulated trans-sialidase that contains the same catalytic site but lacks the tandem amino acid repeats typical of trypomastigote trans-sialidase.
Biologicals, 2013
Rabies is a widespread zoonotic disease responsible for approximately 55,000 human deaths/year. T... more Rabies is a widespread zoonotic disease responsible for approximately 55,000 human deaths/year. The direct fluorescent antibody test (DFAT) and the mouse inoculation test (MIT) used for rabies diagnosis, have high sensitivity and specificity, but are expensive and time-consuming. These disadvantages and the identification of new strains of the virus encourage the use of new techniques that are rapid, sensitive, specific and economical for the detection and research of the Rabies Virus (RABV). Real-time RT-PCR, phylogeographic analysis, proteomic assays and DNA recombinant technology have been used in research laboratories. Together, these techniques are effective on samples with low virus titers in the study of molecular epidemiology or in the identification of new disease markers, thus improving the performance of biological assays. In this context, modern advances in molecular technology are now beginning to complement more traditional approaches and promise to revolutionize the diagnosis of rabies. This brief review presents some of the recent molecular tools used for RABV analysis, with emphasis on rabies diagnosis and research.
Journal of infection in developing countries, 2015
In Brazil, various isolates of rabies virus (RABV) show antigenic profiles distinct from those es... more 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. 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. 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-hematophagou...
The journal of venomous animals and toxins including tropical diseases, 2015
Rabies is an incurable neglected zoonosis with worldwide distribution characterized as a lethal p... more 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. 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 ...
The National Vaccination Rabies Prophylaxis Program aims mainly the maintenance of the protective... more The National Vaccination Rabies Prophylaxis Program aims mainly the maintenance of the protective immunogenic levels in vaccinated animals. The rabies antibody titers are expected in ≥ 0.5 IU / mL, considered by the World Health Organization (WHO) and the Office International des Epizooties (OIE) a reference titration status of protection against rabies virus and an indicator to evaluate the efficacy of the vaccine. The aim of this study was to evaluate the immune response of domestic dogs that received a single dose of rabies cell culture vaccine, considering their age, race and period between the vaccination and the blood collection. To evaluate the immune response in dogs, 432 serum samples received at the Pasteur Institute of São Paulo in the triennium 2009-2011 were analyzed. Based on the exam requisitions, samples were selected from animals that received only one dose of vaccine until the date of blood collection. Information about age, race and period between vaccination and ...
Revista de saúde pública, 2010
To evaluate the humoral immune response to the pre-exposure schedule of human rabies vaccination ... more 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. 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 seroposi...
Revista do Instituto de Medicina Tropical de São Paulo, 1999
An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rab... more An indirect ELISA for determination of post-vaccination rabies antibody was applied. Purified rabies virus was used as antigen to coat plates, and staphylococcal protein A linked with horseradish peroxidase was used for detecting IgG antibody in human sera. Sera from humans, vaccinated with cell-culture vaccine or suckling-mouse-brain vaccine, were examined. ELISA results were compared to those obtained from the virus neutralization test. The mean and standard deviation of OD were determined for 126 negative sera (pre-vaccination) and for 73 sera from vaccinated persons showing antibody titers lower than 0.5 IU/ml. Results were defined as ELISA -positive, -negative or -doubtful. Establishment of a doubtful region reduced the number of sera otherwise classified as positive (false-positive sera). In this way, the sensitivity, specificity and agreement values were respectively 87.5%, 92.4% and 88.5%. No significant differences were observed in these values when the group vaccinated with cell-culture vaccine and the group vaccinated with suckling-mouse-brain vaccine were compared. It was shown that much of the disagreement between the values obtained by neutralization test and ELISA occurred in sera obtained at the beginning of the immunization process, and was probably due to the presence of IgM in the serum samples, detected only by the former test. This ELISA method can be used as a screening test in rabies laboratories regardless of the kind of vaccine used for immunization.
Vaccine, 2007
The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process ... more The immunogenicity and safety of a new human rabies vaccine, produced in Vero cells by a process that does not require supplementation with human or animal derived components in production, were assessed. Thus, the objective is to produce a safer vaccine at a lower cost. A total of 296 volunteers was divided into two groups: Group 1, which received the study vaccine, and Group 2, which received the Vero cells vaccine produced by Sanofi Pasteur. Five doses were given on days 0, 3, 7, 14 and 28. Blood samples for determination of rabies virus neutralizing antibodies were collected on days 0, 14, 38 and 90. The geometric mean titers (GMT) were much higher than 0.5 IU/ml in both groups on days 14, 38 and 90, indicating seroconversion according to the World Health Organization. In Group 1, however, the GMTs were higher than in Group 2, the difference being statistically significant in the two last samples. There was no statistical difference between the groups in the ratio of individuals with titers > or =0.5 IU/ml in each sample. Pain at the injection site was the most common adverse reaction and occurred most often in Group 1 (p < 0.001). All cases had a favorable evolution. There were no severe adverse reactions. It was concluded that the new vaccine is safe and immunogenic.
Revista do Instituto de Medicina Tropical de São Paulo, 1995
Revista de Saúde Pública, 2010
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Papers by Luciana B Chaves