Publications (21)36.94 Total impact
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Article: Antigenic and genetic characterization of rabies virus isolates from uruguay.
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ABSTRACT: After 25 years without any reported cases of rabies in Uruguay, the northern region of the country experienced an epizootic of bovine paralytic rabies in October 2007. The outbreak affected bovines and equines, and the main source of infection was the bat Desmodus rotundus, the only hematophagous species in the country. From October 2007 to July 2008, 42 bovine, 3 equine and 120 chiropteran samples were submitted to the National Veterinary Diagnostic Laboratory for rabies testing. A total of 12 samples (7 bovine, 2 equine and 3 from D. rotundus) were positive by the fluorescent antibody test, and viruses were isolated by the mouse inoculation test. The objective of this study was to compare the antigenic and genetic characteristics of these isolates and three isolates from insectivorous bats from other regions. Antigenic typing using a panel of eight monoclonal antibodies identified all 12 viruses as variant 3 (AgV3), a variant associated with D. rotundus. Two isolates from insectivorous bats (Tadarida brasiliensis and Molossus sp) were characterized as antigenic variant 4 (AgV4) while the third, from Myotis sp., could not be characterized using this panel as its reactivity pattern did not match that of any of the known antigenic variants. Partial N-gene sequences (nt 149-1420) of these isolates were aligned with homologous sequences derived from GenBank by the CLUSTAL/W method and used to build a neighbor-joining distance tree with the Kimura 2-parameter model. All 12 isolates were genetically grouped into the D. rotundus cluster as they shared 100% identity. In the phylogenetic analysis, the three isolates from insectivorous bats segregated into three clusters: one related to Tadarida brasiliensis, one to Myotis sp. and the other to Lasiurus sp., although the isolate associated with the latter came from a Molossus sp specimen. These results indicate that AgV3 was associated with the outbreak of bovine paralytic rabies in Uruguay. This is the first report of rabies virus having been detected in non-hematophagous bats in this country.Virus Research 01/2013; · 2.94 Impact Factor -
Article: Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.
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ABSTRACT: In Brazil, bats have been assigned an increasing importance in public health as they are important rabies reservoirs. Phylogenetic studies have shown that rabies virus (RABV) strains from frugivorous bats Artibeus spp. are closely associated to those from the vampire bat Desmodus rotundus, but little is known about the molecular diversity of RABV in Artibeus spp. The N and G genes of RABV isolated from Artibeus spp. and cattle infected by D. rotundus were sequenced, and phylogenetic trees were constructed. The N gene nucleotides tree showed three clusters: one for D. rotundus and two for Artibeus spp. Regarding putative N amino acid-trees, two clusters were formed, one for D. rotundus and another for Artibeus spp. RABV G gene phylogeny supported the distinction between D. rotundus and Artibeus spp. strains. These results show the intricate host relationship of RABV's evolutionary history, and are invaluable for the determination of RABV infection sources.The Brazilian journal of infectious diseases: an official publication of the Brazilian Society of Infectious Diseases 11/2012; · 0.55 Impact Factor -
Article: Novel monoclonal antibodies that bind to wild and fixed rabies virus strains.
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ABSTRACT: Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.Journal of virological methods 07/2011; 175(1):66-73. · 2.13 Impact Factor -
Article: Antemortem diagnosis of human rabies in a veterinarian infected when handling a herbivore in Minas Gerais, Brazil.
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ABSTRACT: The Ministry of Health's National Human Rabies Control Program advocates pre-exposure prophylaxis (PEP) for professionals involved with animals that are at risk of contracting rabies. We report an antemortem and postmortem diagnosis of rabies in a veterinarian who became infected when handling herbivores with rabies. The antemortem diagnosis was carried out with a saliva sample and a biopsy of hair follicles using molecular biology techniques, while the postmortem diagnosis used a brain sample and conventional techniques. The veterinarian had collected samples to diagnose rabies in suspect herbivores (bovines and caprines) that were subsequently confirmed to be positive in laboratory tests. After onset of classic rabies symptoms, saliva and hair follicles were collected and used for antemortem diagnostic tests and found to be positive by RT-PCR. Genetic sequencing showed that the infection was caused by variant 3 (Desmodus rotundus), a finding confirmed by tests on the brain sample. It is essential that professionals who are at risk of infection by the rabies virus undergo pre-exposure prophylaxis. This study also confirms that molecular biology techniques were used successfully for antemortem diagnosis and therefore not only allow therapeutic methods to be developed, but also enable the source of infection in human rabies cases to be identified accurately and quickly.Revista do Instituto de Medicina Tropical de São Paulo 02/2011; 53(1):39-44. · 1.00 Impact Factor -
Article: Human rabies transmitted by vampire bats: antigenic and genetic characterization of rabies virus isolates from the Amazon region (Brazil and Ecuador).
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ABSTRACT: Since 2004, the main transmitter of human rabies in Latin America has been the vampire bat (Desmodus rotundus). Based on the nucleoprotein of the rabies virus (RV), we analyzed antigenic and genetic profiles of isolates from 29 samples taken from humans living in different areas of the Amazon region. Two isolates were from Ecuador and 27 from the Northern and Northeastern regions of Brazil, which were obtained during outbreaks in various municipalities in the states of Pará and Maranhão in the years 2004 and 2005. The partial N gene (nt 104-1477) of the 29 isolates was sequenced, and the sequences were used to build a neighbor-joining tree with the Kimura-2 parameter model. All 29 human RV isolates were identified as belonging to antigenic variant 3 (AgV3) and were genetically grouped into the D. rotundus cluster, which was divided into two subclusters (A and B), subcluster A in turn being divided into four genetic groups (A1, A2, A3 and A4). Genetic and molecular markers characterizing these genetic lineages were also identified. The results of this study show that the isolates belong to the same rabies cycle as that of the vampire bat D. rotundus. However, the division of clusters within the lineage associated with D. rotundus shows that different genetic sublineages of the virus were circulating in the Amazon region during the study period. Our findings suggest that there are phylogeographic differences between isolates obtained over a short period.Virus Research 10/2010; 153(1):100-5. · 2.94 Impact Factor -
Article: Rabies virus in insectivorous bats: implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology.
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ABSTRACT: Insectivorous bats are the main reservoirs of rabies virus (RABV) in various regions of the world. The aims of this study were to (a) establish genealogies for RABV strains from different species of Brazilian insectivorous bats based on the nucleoprotein (N) and glycoprotein (G) genes, (b) investigate specific RABV lineages associated with certain genera of bats and (c) identify molecular markers that can distinguish between these lineages. The genealogic analysis of N and G from 57 RABV strains revealed seven genus-specific clusters related to the insectivorous bats Myotis, Eptesicus, Nyctinomops, Molossus, Tadarida, Histiotus and Lasiurus. Molecular markers in the amino acid sequences were identified which were specific to the seven clusters. These results, which constitute a novel finding for this pathogen, show that there are at least seven independent epidemiological rabies cycles maintained by seven genera of insectivorous bats in Brazil.Virology 09/2010; 405(2):352-60. · 3.35 Impact Factor -
Article: Genetic characterization of rabies virus isolated from bovines and equines between 2007 and 2008, in the States of São Paulo and Minas Gerais.
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ABSTRACT: Rabies is an acute disease of the central nervous system and is responsible for the deaths of thousands of humans, wild animals and livestock, particularly cattle, as well as causing major economic losses. This study describes the genetic characterization of rabies virus variants that circulate in Desmodus rotundus populations and are transmitted to herbivores. Fifty rabies virus isolates from bovines and equines in the States of São Paulo and Minas Gerais, Brazil, were genetically characterized and compared with sequences retrieved from GenBank. Two clusters (I and II) with mean nucleotide identities of 99.1 and 97.6% were found. The first of these contained nearly all the samples analyzed. Lineages from other Brazilian states grouped in cluster II. Analysis of the amino acid sequences of the N proteins revealed the existence of genetic markers that may indicate possible variations between geographic regions, although the biologically active regions are conserved within the species over space and time.Revista da Sociedade Brasileira de Medicina Tropical 04/2010; 43(2):116-20. · 0.68 Impact Factor -
Article: Molecular characterization of Rabies Virus isolates from dogs and crab-eating foxes in Northeastern Brazil.
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ABSTRACT: Thirty-eight samples of Rabies Virus isolated from dogs and crab-eating foxes (Cerdocyon thous) in Northeastern Brazil were characterized genetically by analyzing the G gene and the psi region. The results show that there are two groups of Rabies Virus lineages circulating among domestic and wild animals in the region. The topologies of the phylogenetic trees of the G gene and psi region are similar and reveal the existence of geographic groups. The genetic diversity of the lineages isolated from wild animals (wild group) was approximately twice that of the lineages isolated from domestic animals (domestic group), and the genetic distance between the two groups was 9.93%. Polymorphism analysis revealed specific intra- and inter-group molecular signatures for both the G gene and psi region. Together with the analysis of the N gene undertaken previously, the results of this study confirm the existence of a Rabies Virus phylogroup in Northeastern Brazil (NB) circulating in the C. thous population, making this species a rabies biotype in the region.Virus Research 02/2009; 141(1):81-9. · 2.94 Impact Factor -
Article: First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil.
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ABSTRACT: 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.Journal of Clinical Laboratory Analysis 02/2009; 23(1):7-13. · 1.38 Impact Factor -
Article: Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA.
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ABSTRACT: Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.The Brazilian journal of infectious diseases: an official publication of the Brazilian Society of Infectious Diseases 01/2009; 12(6):462-5. · 0.55 Impact Factor -
Article: Characterization of Rabies virus isolated from canids and identification of the main wild canid host in Northeastern Brazil.
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ABSTRACT: The rabies cases in dogs and wild canids in Northeastern Brazil are a public and animal health problem. This paper describes the identities of the coding region of the N-gene of Rabies virus (RABV) isolated in canids from Northeastern Brazil. The genetic tree generated using the sequence data described here divided the cluster BRAZILAN CANIDS into two subclusters (DOG-RELATED STRAINS and WILD CANID-RELATED STRAINS) with identities greater than those already described. The two subclusters are sub-divided into geographic groups related to the origin of the isolates, suggesting a long-standing ecological coexistence of the sequence types characteristic of the groups. This article also analyzes the 513-nucleotide stretch of the mitochondrial DNA control region of rabies-positive canids from Northeastern Brazil with a view to identifying the main RABV host among them. Among the four species of wild canids found in the region, two (Cerdocyon thous and Pseudalopex vetulus) are frequently associated with rabies. Phylogenetic analysis of sequence data generated from mtDNA suggests that C. thous is the main wild canid host in the region. The results obtained in this study are in concordance with the zoology and ecology of wild canids, and thus, help improve epidemiologic vigilance of rabies and allow a more targeted control of the disease.Virus Research 02/2008; 131(1):33-46. · 2.94 Impact Factor -
Article: [Rabies virus in naturally infected bats in the State of São Paulo, Southeastern Brazil].
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ABSTRACT: To identify the species of bats involved in maintaining the rabies cycle; to investigate the distribution of the rabies virus in the tissues and organs of bats and the time taken for mortality among inoculated mice. From April 2002 to November 2003, bats from municipalities in the State of São Paulo were screened for the presence of the rabies virus, by means of direct immunofluorescence. The virus distribution in the bats was evaluated by inoculating mice and N2A cells with 20% suspensions prepared from fragments of different organs and tissues, plus the brain and salivary glands. The time taken for mortality among the mice was monitored daily, following intracerebral inoculation. Out of the 4,395 bats received, 1.9% were found positive for the rabies virus. They belonged to ten genera, with predominance of insectivores. The maximum mean times taken for mortality among the mice following inoculation with brain and salivary gland material were 15.33+/-2.08 days and 11.33+/-2.30 days for vampire bats, 16.45+/-4.48 days and 18.91+/-6.12 days for insectivorous bats, and 12.60+/-2.13 days and 15.67+/-4.82 days for frugivorous bats, respectively. The species infected with the rabies virus were: Artibeus lituratus, Artibeus sp., Myotis nigricans, Myotis sp., Eptesicus sp., Lasiurus ega, Lasiurus cinereus, Nyctinomops laticaudatus, Tadarida brasiliensis, Histiotus velatus, Molossus rufus, Eumops sp. and Desmodus rotundus. Virus investigation in the different tissues and organs showed that the brain and salivary glands were the most suitable sites for virus isolation.Revista de Saúde Pública 07/2007; 41(3):389-95. · 1.33 Impact Factor -
Article: Short-interfering RNAs as antivirals against rabies.
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ABSTRACT: This study aimed to test in vitro a RNA-interference based antiviral approach for rabies with short-interfering RNAs (siRNAs) against rabies virus nucleoprotein mRNA. BHK-21 cells were infected with serial dilutions of PV rabies virus strain and transfected with a pool of three siRNAs. Direct immunofluorescence staining showed a 5-time decrease in virus titer when compared to a non-treated plate, showing a promising new approach to the development of antivirals for rabies treatment.Brazilian Journal of Infectious Diseases 05/2007; 11(2):224-5. · 1.00 Impact Factor -
Article: Padronização e aplicação da técnica de isolamento do vírus da raiva em células de neuroblastoma de camundongos (N2A)
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ABSTRACT: Rabies laboratorial diagnosis is very important since clinical diagnosis is not precise. Fluorescent Antibody Test (FAT) is the most used test and even though it is highly sensible, accurate and fast, false negatives results may occur. Thus, the isolation of rabies virus in mice (VIM) of Central Nervous System (CNS) samples of suspected animals to be infected is also recommended and, nowadays, this test has been substituted in many laboratories by viral isolation in cell culture (VICC). The aim of the present study was to compare the sensibility of virus isolation in murine neuroblastoma (N2A) cell culture with VIM test and with FAT, as well as evaluate obtained results in the diagnostic routine from Pasteur Institute, regarding reduction of costs, time and work. A total of 105 CNS samples of different animal species were analyzed by FAT, VIM and VICC: 50 bats, 32 dogs, 13 foxes and 10 bovines. All bats and bovines samples presented concordant results for the three tests, while dogs and foxes samples presented concordance only in 24 samples (69%). Based on these results, since 2004 it has been instituted that all bat samples sent to Pasteur Institute Laboratory, after diagnosed by FAT, should be submitted to viral isolation in cell culture, replacing the use of mice. In the period of January 2004 to September 2007, 11.298 bat samples were analyzed. A total of 67 positive samples for IFD and/or VICC were also submitted to VIM, and 61 samples presented concordant results for the three tests, and showed that the use of N2A cells is more sensible to “street virus” isolation of bat samples in laboratorial routine, being faster and lower in costs than VIMBoletim Epidemiológico Paulista. 01/2007; 4(47):12-18. -
Article: Reservatórios silvestres do vírus da raiva: um desafio para a saúde pública
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ABSTRACT: Human mortality from endemic canine rabies is estimated to be 55.000 deaths per year, especially in Asia and Africa. In Latin America the canine rabies was endemic until 1980, but recently had a frequency reduction of canine rabies virus and consequently in human. With the development of “Plan of Action for the Elimination of Urban Rabies” the wildlife animals has become epidemiologically important and can be a new challenge. The most common transmitters of rabies to humans were a vampire-bat Desmodus rotundus in the period 2004-2005 (68%). Besides of the three species of vampire-bats, whose the rabies virus were isolated, there are 33 other species of non-hematophagous-bat that were related infected with the same virus. Bats (order Chiroptera) and canids (order Carnivora) are recognized as wildlife reservoirs. In Northeastern Brazil, the disease have had high fre- quency in Cerdocyon thous (crab-eating fox) in parallel with another epidemiological cycle rabies virus in Callithrix jacchus (marmoset) which the disease distribution is unknown. The authors describe the disease's characteristics in bats and canids, the control strategies and emphasize the importance of antigenic and genetics studies as tool of epidemiological surveillanceBoletim Epidemiológico Paulista. 01/2007; 4(40):2-8. -
Article: Laboratory diagnosis of equine rabies and its implications for human postexposure prophylaxis.
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ABSTRACT: Laboratory diagnosis is essential to confirm suspected cases of equine rabies and to determine the medical care needed for human postexposure antirabies prophylaxis. Equine rabies transmitted by the vampire bat, Desmodus rotundus, has increased gradually in the State of São Paulo. The present study has several objectives, the most important being the evaluation of fluorescent antibody test (FAT) and virus-isolation laboratory tests performed with different equine nervous system tissues (cortical, hippocampus, cerebellar, brainstem and cervical medullar) to determine the tissue for which the two techniques have the highest sensitivity. Analysis by FAT of these five regions of the central nervous system (CNS) from 35 animals showed that there was a greater amount of viral antigen in the brainstem and cervical medullar tissues than in the hippocampus, cortical and cerebellar tissues. While there were no significant differences in the mortality rate of mice inoculated with suspension prepared from the different tissues, a trend towards higher mortality rate was detected with brainstem and cervical medullar tissues. Laboratory diagnosis was not affected by whether the animal had been vaccinated or not, or whether it had died following the natural course of the disease or as a result of euthanasia. Isolation of the rabies virus in equine salivary glands demonstrated the potential risk for humans exposed to infected animals.Journal of Virological Methods 01/2007; 138(1-2):1-9. · 2.01 Impact Factor -
Article: Diagnosis of human rabies cases by polymerase chain reaction of neck-skin samples.
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ABSTRACT: Rapid diagnosis of rabies in suspected human cases influences post-exposure prophylaxis for potential contacts of the patient and ensures appropriate patient management. Apart from the central nervous system (CNS), rabies virus (RABV) is usually present in small sensory nerves adjacent to hair follicles of infected humans. We used an RT-PCR, with primers targeted to the 3' terminal portion of the nucleoprotein gene (N), to test neck-skin samples of nine patients who had rabies in order to validate a diagnostic method that could serve as an additional tool for rabies diagnosis, particularly in antemortem samples. Six of eight postmortem samples were found to be positive for rabies by RT-PCR, and one of two samples collected antemortem was positive with this same technique. Results were confirmed by DNA sequencing; this validates RT-PCR and neck-skin as a suitable technique and type of sample, respectively, for use in the diagnosis of human rabies. RT-PCR applied to neck-skin biopsies could allow early diagnosis and lead to more effective rabies treatment.Brazilian Journal of Infectious Diseases 11/2006; 10(5):341-5. · 1.00 Impact Factor -
Article: Postmortem confirmation of human rabies source.
Emerging infectious diseases 06/2006; 12(5):867-9. · 6.17 Impact Factor -
Article: Flow cytometry assay for intracellular rabies virus detection.
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ABSTRACT: Following previous studies reporting microbiological diagnosis by flow cytometry, the possibility of using this method was examined to monitor infection of susceptible cell lines by a fixed rabies virus strain (Pasteur Virus strain-PV) or a wild rabies virus strain (WRS). Suspensions of BHK-21 and C6 cells were infected with viruses and a time course of virus infection was established. Sequentially, at several time points, infected and control uninfected cells were fixed, permeabilized, and stained with a rabies virus-specific antibody conjugate. This was achieved by resuspending cells in a solution containing p-formaldehyde in FACS lysis fluid, which allowed the detection of intracellular virus with flourescein-coupled antibodies by flow cytometry. A Becton Dickinson FACSCalibur flow cytometer was used to analyze the percentage of cells infected and the kinetics of the infection process was determined. As early as 12 h after inoculation with both rabies virus strains, significant levels (P<0.01) of infection (from 4.7 to 7.1%) were detected by flow cytometry. The maximum level of infection was obtained at 48 h in C6 cells (88%) with both viruses. The advantages of this method for examination of intracellular virus infection are discussed.Journal of Virological Methods 08/2002; 105(1):181-6. · 2.01 Impact Factor -
Article: Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil.
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ABSTRACT: Although the main transmitters of rabies in Brazil are dogs and vampire bats, the role of other species such as insectivorous and frugivorous bats deserves special attention, as the rabies virus has been isolated from 36 bat species. This study describes the first isolation of the rabies virus from the insectivorous bat Eumops perotis. The infected animal was found in the city of Ribeirão Preto, São Paulo. The virus was identified by immunofluorescence antibody test (FAT) in central nervous system (CNS) samples, and the isolation was carried out in N2A cell culture and adult mice. The sample was submitted to antigenic typing using a panel of monoclonal antibodies (CDC/Atlanta/USA). The DNA sequence of the nucleoprotein gene located between nucleotides 102 and 1385 was aligned with homologous sequences from GenBank using the CLUSTAL/W method, and the alignment was used to build a neighbor-joining distance-based phylogenetic tree with the K-2-P model. CNS was negative by FAT, and only one mouse died after inoculation with a suspension from the bat's CNS. Antigenic typing gave a result that was not compatible with the patterns defined by the panel. Phylogenetic analysis showed that the virus isolated segregated into the same cluster related to other viruses isolated from insectivorous bats belonging to genus Nyctinomops ssp. (98.8% nucleotide identity with each other).Revista do Instituto de Medicina Tropical de São Paulo 50(2):95-9. · 1.00 Impact Factor
Top co-authors
Top Journals
Institutions
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2013
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Ministry of Livestock, Agriculture and Fisheries
Montevideo, Departamento de Montevideo, Uruguay
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2007–2008
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Ministério da Saúde do Brasil
São Paulo, Estado de Sao Paulo, Brazil -
Universidade de São Paulo
- Departamento de Medicina Veterinária (ZMV) (Pirassununga)
Ribeirão Preto, Estado de Sao Paulo, Brazil
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2006
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Instituto Evandro Chagas
Belém, Estado do Para, Brazil
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