Eastern equine encephalitis cases among horses in Brazil between 2005 and 2009.

Eastern equine encephalitis is a viral zoonosis that exhibits complex distribution and epidemiology, and greater importance should be given to this disease by the public-health authorities. In Brazil, although eastern equine encephalitis virus (EEEV) has been identified in vectors and antibodies are sometimes detected in horses and humans, there have been no records of equine encephalitis in horses caused by this virus during the last 24 years. This study describes eighteen cases of eastern equine encephalomyelitis that occurred in six Brazilian states between 2005 and 2009. Viral RNA was identified using semi-nested RT-PCR to detect members of the genus Alphavirus, and by genetic sequencing. The gene encoding NSP1 was partially amplified, and after genetic sequencing, eighteen sequences were generated. All eighteen strains were classified as belonging to lineage III of American EEEV. These findings could be an indication of the importance of this virus in animal and human public health.

No Evidence of Rabies Exposure in Wild Marmosets (Callithrix jacchus) of Northeast Brazil.

Rabies transmitted by wildlife is the main source of human rabies mortality in Latin America and considered an emerging disease. The common marmoset Callithrix jacchus of Brazil is the only known primate reservoir of rabies worldwide. We tested whether alive free-ranging C. jacchus were exposed to rabies in four northeast states that have previously reported rabies-positive dead C. jacchus (Pernambuco and Bahia) or not (Paraíba and Rio Grande do Norte). Our results show no evidence of rabies antibodies or infection in the sampled C. jacchus, suggesting that apparently healthy marmosets are not widely exposed to rabies over their natural range.

Spatio-temporal dynamics of rabies and habitat suitability of the common marmoset Callithrix jacchus in Brazil.

Rabies transmitted by wildlife is now the main source of human rabies in the Americas. The common marmoset, Callithrix jacchus, is considered a reservoir of rabies causing sporadic and unpredictable human deaths in Brazil, but the extent of the spillover risk to humans remains unknown. In this study, we described the spatiotemporal dynamics of rabies affecting C. jacchus reported to Brazil's Ministry of Health passive surveillance system between 2008 and 2020, and combined ecological niche modelling with C. jacchus occurrence data to predict its suitable habitat. Our results show that 67 outbreaks (91 cases) of rabies affecting C. jacchus were reported by 41 municipalities between January 2008 and October 2020, with a mean of 5 outbreaks/year [range: 1-14]. The maximum number of outbreaks and municipalities reporting cases occurred in 2018, coinciding with higher surveillance of primate deaths due to Yellow Fever. A mean of 3 [1-9] new municipalities reported outbreaks yearly, suggesting potential spatial expansions of the C. jacchus variant in northeastern Brazil and emerging rabies spillover from vampire bat Desmodus rotundus to C. jacchus in the north and south. Outbreaks were concentrated in the states of Ceará (72%) and Pernambuco (16%) up to 2012, but are now reported in Piauí since 2013, in Bahia since 2017 (D. rotundus' antigenic variant, AgV3) and in Rio de Janeiro since 2019 (AgV3). Besides confirming suitable habitat for this primate in the northeast and the east coast of Brazil, our Maximum Entropy model also predicted suitable habitat on the north and the west states of the country but predicted low habitat suitability among inland municipalities of the Caatinga biome reporting rabies. Our findings revealed new areas reporting rabies infecting C. jacchus, highlighting the need to implement strategies limiting spillover to humans and to better understand the drivers of C. jacchus rabies dynamics.

Molecular characterization of Rabies Virus isolates from dogs and crab-eating foxes in Northeastern Brazil.

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.

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.

Non-human primates as a reservoir for rabies virus in Brazil.

Rabies virus (RABV) does not persist in the environment as it is a very fragile agent. The primary hosts are mammalian species in the orders Carnivora and Chiroptera. Since the late 1980s, RABV has been isolated from non-human primates, Callithrix jacchus (the white-tufted marmoset), in four coastal states (Rio Grande do Norte, Ceará, Piauí and Pernambuco) in north-eastern Brazil, where this species is indigenous. The original habitat of C. jacchus consisted of two Brazilian biomes, the Atlantic Forest and the Caatinga. However, these marmosets have since adapted to other ecosystems as a result of human activities. Between 1988 and 1989, RABV isolates were obtained from white-tufted marmosets in the state of Rio Grande do Norte, but antigenic and genetic identification studies were not conducted at that time. In the following years, three additional states reported cases (Ceará, Piauí and Pernambuco). In two of these states (Ceará and Piauí), human cases of rabies transmitted by marmosets were reported. According to Brazilian Health Ministry data, at least 19 human cases in which this species was the source of infection were registered in between 1990 and 2016. Recent findings in laboratory tests of 12 rabid samples from humans and marmosets and the regional transmission among these animals for over 20 years, together with the gradual increase in the affected geographic area, support the concept of the emergence of a new RABV reservoir. Regional tourism, the wild animal trade and the cultural practice of maintaining these animals as pets, particularly in coastal regions, appear to be major risk factors for the increase in human cases. Additional epidemiological and ecological studies are required to better understand local disease dynamics and to identify ideal opportunities for prevention and control of this fatal infection.

Characterization of Rabies virus isolated from canids and identification of the main wild canid host in Northeastern Brazil.

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.

Antigenic and genetic characterization of the first rabies virus isolated from the bat Eumops perotis in Brazil.

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).

Short-interfering RNAs as antivirals against rabies.

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.

[Rabies virus in naturally infected bats in the State of São Paulo, Southeastern Brazil]. / Vírus da raiva em quirópteros naturalmente infectados no Estado de São Paulo, Brasil.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Laboratory diagnosis of equine rabies and its implications for human postexposure prophylaxis.

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.

Diagnosis of human rabies cases by polymerase chain reaction of neck-skin samples.

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.

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.

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil.

Rabies virus (RABV) isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2), Desmodus rotundus (variant 3), crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT) and real time polymerase chain reaction (qRT-PCR) for quantification of rabies virus nucleoprotein gene (N gene). Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

Flow cytometry assay for intracellular rabies virus detection.

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.

Antigenic typing of Brazilian rabies virus samples isolated from animals and humans, 1989-2000.

Animal and human rabies samples isolated between 1989 and 2000 were typified by means of a monoclonal antibody panel against the viral nucleoprotein. The panel had been previously established to study the molecular epidemiology of rabies virus in the Americas. Samples were isolated in the Diagnostic Laboratory of the Pasteur Institute and in other rabies diagnostic centers in Brazil. In addition to the fixed virus samples CVS-31/96-IP, preserved in mouse brain, and PV-BHK/97, preserved in cell culture, a total of 330 rabies virus samples were isolated from dogs, cats, cattle, horses, bats, sheep, goat, swine, foxes, marmosets, coati and humans. Six antigenic variants that were compatible with the pre-established monoclonal antibodies panel were defined: numbers 2 (dog), 3 (Desmodus rotundus), 4 (Tadarida brasiliensis), 5 (vampire bat from Venezuela), 6 (Lasiurus cinereus) and Lab (reacted to all used antibodies). Six unknown profiles, not compatible with the panel, were also found. Samples isolated from insectivore bats showed the greatest variability and the most commonly isolated variant was variant-3 (Desmodus rotundus). These findings may be related to the existence of multiple independent transmission cycles, involving different bat species.

Antigenic and genetic characterization of rabies virus isolates from Uruguay.

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 T. 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.

Desmodus rotundus and Artibeus spp. bats might present distinct rabies virus lineages.

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.

Novel monoclonal antibodies that bind to wild and fixed rabies virus strains.

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.

Antemortem diagnosis of human rabies in a veterinarian infected when handling a herbivore in Minas Gerais, Brazil.

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.