Rafael de Novaes Oliveira

A coronavirus detected in the vampire bat Desmodus rotundus

This article reports on the identification of a group 2 coronavirus (BatCoV DR/2007) in a Desmodus rotundus vampire bat in Brazil. Phylogenetic analysis of ORF1b revealed that BatCoV DR/2007 originates from a unique lineage in the archetypical group 2 coronaviruses, as described for bat species elsewhere with putative importance in Public Health.

Outbreaks of Eastern equine encephalitis in northeastern Brazil

Outbreaks of eastern equine encephalitis observed from May 2008 to August 2009 in the Brazilian states of Pernambuco, Ceará, and Paraíba are reported. The disease occurred in 93 farms affecting 229 equids with a case fatality rate of 72.92%. Main clinical signs were circling, depression or hyperexcitability, ataxia, and progressive paralysis with a clinical manifestation period of 3–15 days. Main histologic lesions were a diffuse lymphocytic encephalomyelitis with neuronal death, satellitosis, neuronophagia, and hemorrhages being more severe in the cerebral gray matter of the telencephalon, diencephalon, and mesencephalon. Some animals also had areas of malacia in the telencephalon, thalamus, and basal nuclei. From 1 case, the virus was isolated by mice inoculation, and in other 13 cases was identified as Eastern equine encephalitis virus by semi-nested reverse transcription polymerase chain reaction. After DNA sequencing, all samples were identified as eastern equine encephalitis through the BLASTn analysis, but samples from the Ceará and Paraíba states corresponded to the same cluster, while the sample from the state of Pernambuco corresponded to a different cluster.

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.

Human rabies transmitted by vampire bats: Antigenic and genetic characterization of rabies virus isolates from the Amazon region (Brazil and Ecuador)

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.

Phylogenetic and epidemiologic evidence of multiyear incubation in human rabies

Eight years after emigrating from Brazil, an otherwise healthy man developed rabies. An exposure prior to immigration was reported. Genetic analysis revealed a canine rabies virus variant found only in the patients home country, and the patient had not traveled internationally since immigrating to the United States. We describe how epidemiological, phylogenetic, and viral sequencing data provided confirmation that rabies encephalomyelitis may present after a long, multiyear incubation period, a consideration that previously has been hypothesized without the ability to exclude a more recent exposure. Accordingly, rabies should be considered in the diagnosis of any acute encephalitis, myelitis, or encephalomyelitis. ANN NEUROL 2014;75:155–160

Genetic characterization of rabies virus isolated from bovines and equines between 2007 and 2008, in the States of São Paulo and Minas Gerais

INTRODUCTION 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

A Comparative Study of Rabies Virus Isolates from Hematophagous Bats in Brazil

The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur.

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 RABVs evolutionary history, and are invaluable for the determination of RABV infection sources.

Comparative analysis of rabies virus isolates from Brazilian canids and bats based on the G gene and G-L intergenic region

Rabies virus (RABV) isolates from two species of canids and three species of bats were analyzed by comparing the C-terminal region of the G gene and the G-L intergenic region of the virus genome. Intercluster identities for the genetic sequences of the isolates showed both regions to be poorly conserved. Phylogenetic trees were generated by the neighbor-joining and maximum parsimony methods, and the results were found to agree between the two methods for both regions. Putative amino acid sequences obtained from the G gene were also analyzed, and genetic markers were identified. Our results suggest that different genetic lineages of RABV are adapted to different animal species in Brazil.

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.