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.

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.

Failure of protection induced by a Brazilian vaccine against Brazilian wild rabies viruses

This report shows that the SMB vaccine currently used in Brazil for human immunisation provides different degrees of protection in mice, depending on the rabies virus strain used as challenge. Using the NIH and Habel potency tests to evaluate the protective activity of rabies vaccine, we observed that vaccinated mice showed a higher resistance to a challenge with a fixed rabies virus (CVS-Challenge Virus Strain). The vaccine potency using the Habel or NIH tests was respectively > 6.4 (log 10) and 1.0 (Relative Potency-RP) when the fixed rabies virus was used for challenge, and from 2.9 to 4.3 (log 10) or 0.13 to 0.8 (RP) when different wild rabies viruses were used for challenge. The presence of virus neutralising antibodies (VNA) could not explain the differences of susceptibility after vaccination, since sera of vaccinated animals had similar VNA levels against both fixed and wild strains before virus challenge (respectively, 5.6 +/- 0.24 and 5.0 +/- 0.25 IU/ml of VNA against the fixed rabies virus and the 566-M strain of wild rabies virus in sera of mice vaccinated with 0.2 units of vaccine). Only cell-mediated immunity parameters correlated with the protection induced by vaccination. The IFN gamma titers found in sera and brain tissues of animals challenged with CVS strain were higher (from 36.7 +/- 5.7 to 293.3 +/- 46.2 IU/ml) than those found in mice challenged with 566-M virus strain (from 16.7 +/- 5.8 to 36.7 +/- 5.8). The proliferation index of spleen cells obtained with CVS stimulation reached a maximal value of 15.1 +/- 0.7 while spleen cells from vaccinated mice stimulated with 566-M virus failed to proliferate. The implications of these data in human protection by vaccination are discussed.

Effect of vaccination and the immunomodulators "bacillus of Calmette-Guerin, Avridine and Propionibacterium acnes" on rabies in mice

Responses of vaccination and treatment to immunomodulators against rabies in mice were evaluated through macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of g-interferon (IFN-g). Onco-BCG, Avridine and Propionibacterium acnes were administered to groups of mice. Higher survival rates were found in animals treated with P. acnes. Lower levels of IFN-g were observed in the groups of infected and vaccinated mice. The IPI was not effective on detecting the response of delayed-type hypersensitivity. Vaccine induced in the infected animals a more intense response to MIF reaction.