Molecular epidemiology of rabies virus strains isolated from wild canids in Northeastern Brazil

Rabies in wild canids in Northeastern Brazil is frequent and has been reported for some time, with episodes of rabies transmission from these animals to humans also reported. In this study, we analyzed the antigenic and genetic profiles of the rabies virus nucleoprotein gene, isolated from 20 samples taken from domestic animals and wild canids located in the Northeastern region of Brazil. All viruses isolated from domestic animals (dogs and cats) belonged to the antigenic variant 2 (AgV2). Among the wild animal samples, only four were AgV2, and nine showed a divergent antigenic profile. Phylogenetic analysis revealed two Brazilian clusters. Cluster 1 (Brazilian domestic carnivore-related strains) showed two subclusters, called 1A and 1B, and cluster 2 (Brazilian wild canid-related strains) also showed two subclusters, called 2A and 2B. The majority of the samples with divergent antigenic strains segregated into subcluster 2A. The intracluster identity of cluster 1 was 95.6% and that of cluster 2, 92.4%. When clusters 1 and 2 were compared, an identity of 88.6% was found. The genetic analysis of wild canid samples performed in this study indicates that there are two distinct rabies cycles among canids in Brazil, one represented by domestic canids and the other by wild canids. This study shows that the virus samples isolated in Northeastern Brazil are region and species-specific.

Phylogeny of rabies virus identified in a cat closely related to vampire bat rabies based on the nucleoprotein gene

Rabies-infected bats are preyed upon by dogs and cats and the transmission of vírus from bats to domestic animais has been reported. It is expected that bat-related virus variants might be found with higher frequency in dogs and cats living in urban areas where the terrestrial cycle has been controlled. This article reports the genetic characterization of one sample of rabies virus from a cat that had contact with a bat on the border of São Paulo city. The sample was genetically typed as variant 3, associated with Desmodus rotundus, suggesting that bats and their rabies virus variants must from now on be considered in the epidemiology of rabies of urban domestic animais and in public health policies.

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.

Inactivated suckling mouse brain rabies vaccine provides short-term immunity i capuchin monkeys (Cebus Apella)

Eight capuchin monkeys (Cebus apella) were vaccinated against rabies with an inactivated suckling mouse brain vaccine (SMBV). Three 1-ml doses of 2% brain tissue suspension were given by i.m. injection at 0, 30 and 60 days. Blood samples were collected at 0, 30, 90, 150, 210, 240, 300 and 365 days and were tested by simplified fluorescence inhibition to titer-neutralizing antibodies. All of the animals developed neutralizing antibodies with titers > 0,5 UI/ml after vaccination, but the immune response persisted for only 122.3 ± 36.6 days. The SMBV was able to induce immune response in the capuchin monkeys, but protection was short-lived.

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107)

The susceptibility of the C6 rat glioma line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supertants of infected cultures by both BHK-21 cell infection and mice inoculation C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new useful system for rabies virus investigation.