Pedro Carnieli

Rabies virus in insectivorous bats: implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology.

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.

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.

Genetic characterization of Rabies virus isolated from cattle between 1997 and 2002 in an epizootic area in the state of São Paulo, Brazil.

The biogeographical history of rabies can be reconstructed using molecular data. This work describes the genetic characterization of the Rabies virus variant that circulates in the Desmodus rotundus (vampire bat) population in an epizootic area and is transmitted to herbivorous livestock. The N and G genes of this virus were sequenced, and the phylogenetic trees generated were topologically concordant. Three genetic clusters were identified in the epizootic area and were designated RD1, RD2 and RD3. The results show that the origins of the epizootics in areas RD1 and RD2 were different and that the epizootic in area RD3 was the result of expansion of that in area RD2. The two genes analyzed are conserved, and their identities, which are greater than 98%, were maintained over time and space. The genetic sequences in this study were compared with others retrieved from GenBank, and the high identity of the N and G genes was also shown to be maintained over time and space. The results suggest that the D. rotundus lineages of the Rabies virus from the Atlantic coast of South America are highly conserved.

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.

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.

Biotechnology advances: A perspective on the diagnosis and research of Rabies Virus

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.

Phylogeography of rabies virus isolated from herbivores and bats in the Espírito Santo State, Brazil

Rabies is enzootic in the State of Espírito Santo, Brazil. Every year, cattle and horses die from rabies that is transmitted by the vampire bat Desmodus rotundus. This paper describes the spread of the rabies virus by the continuous diffusion model using relaxed random walks with BEAST software. Forty-one (41) sequences of gene G from the rabies virus that was isolated from bats and domestic herbivores from several areas of the state between 2006 and 2010 were analyzed. The phylogenetic tree showed three main clusters as well as two sub-clusters under cluster 2. A spatial analysis showed that three strains of the rabies virus spread independently. In general, central Espírito Santo, which is mountainous, was the area where separation of the virus strains occurred. This physical barrier, however, was overcome at some point in time, as samples from different lineages were found in the same microarea.

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.