Caracterização molecular do vírus da raiva isolado de Desmodus rotundus capturados no Estado do Rio de Janeiro / Molecular characterization of rabies virus isolated from Desmodus rotundus captured in Rio de Janeiro State

Caracterizou-se filogeneticamente o vírus da raiva, isolado de morcegos hematógafos (Demodus rotundus). Cento e noventa e nove D. rotundus foram capturados em cinco abrigos, no Norte e Noroeste do Estado do Rio de Janeiro e sul do Espírito Santo. Sete deles foram positivos para a raiva. Amostras desses vírus foram sequenciadas e comparadas com sequências provenientes de diversos estados brasileiros. As sequências de vírus da raiva isoladas, na região norte do Estado do Rio de Janeiro, mostraram características que as distinguem de amostras de vírus isoladas em outras regiões do país, no entanto foram idênticas às isoladas de bovinos no noroeste do Rio de Janeiro.

Rabies samples isolated from vampire bats captured in the Rio de Janeiro State were phylogenetically analyzed. One hundred and ninety nine vampire bats were captured from five shelters from North and Northwest of Rio de Janeiro and South of Espírito Santo States. Seven of them were positive for rabies. Theses samples were sequenced and compared with rabies virus sequences from several Brazilian states. The sequences of rabies virus, isolated in the present work, from North of Rio de Janeiro State, showed characteristics that differ of the sequences isolated from bats from other Brazilian regions. However, they were identical to samples isolated from cattle in Northwest of Rio de Janeiro state.

Molecular analysis of the N gene of canine distemper virus in dogs in Brazil

Eleven central-nervous-system samples collected from stray dogs between 2000 and 2004 were found positive by RT-PCR, which amplified a 480bp fragment of the N gene of canine distemper virus (CDV). Phylogenetic analysis based on partial N-gene sequences showed four major clusters. All dog strains segregated into cluster I, with a mean nucleotide identity of 95.8 percent and 95.6 percent with the Onderstepoort and Lederle vaccine strains, respectively. Cluster II contained all the raccoon-related strains, cluster III Orient strains and Cluster IV the Onderstepoort and Lederle vaccine strains, with a mean nucleotide identity of 99.7 percent between them. This is the first report of phylogenetic analysis of CDV strains in Brazil.

Onze amostras de sistema nervoso central de cães coletados entre 2000 e 2004 foram positivas pela RT-PCR, a qual amplificou um fragmento de 480pb do gene N do vírus da cinomose canina (VCC). A análise filogenética baseada na seqüência parcial do gene N mostrou quatro principais agrupamentos genéticos. Todas as amostras de cães segregaram no agrupamento I, com identidade média de nucleotídeos de 95,8 por cento e 95,6 por cento com as amostras vacinais Onderstepoort e Lederle, respectivamente. O agrupamento II agregou todas as amostras relacionadas aos guaxinins. O agrupamento III agregou amostras orientais e o agrupamento IV agregou as amostras vacinais Onderstepoort e Lederle, com identidade média de nucleotídeos de 99,7 por cento entre elas. Este é o primeiro relato de análise filogenética de amostras de VCC no Brasil.

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.

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

The susceptibility of the C6 rat glioma cell 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 supernatants 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 and useful system for rabies virus investigation.

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.

Antibody response in cattle after vaccination with inactivated and attenuated rabies vaccines.

Despite the absence of current official reports showing the number of cattle infected by rabies, it is estimated that nearly 30,000 bovines are lost each year in Brazil. In order to minimize the important economic losses, control of the disease is achieved by eliminating bat colonies and by herd vaccination. In this study, we compare the antibody response in cattle elicited by vaccination with an attenuated ERA vaccine (AEvac) and an inactivated-adjuvanted PV (IPVvac) vaccine. The antibody titers were appraised by cell-culture neutralization test and ELISA, and the percentage of seropositivity was ascertained for a period of 180 days. IPVvac elicited complete seropositivity rates from day 30 to day 150, and even on day 180, 87% of the sera showed virus-neutralizing antibody titers (VNA) higher than 0.5IU/ml. There were no significant differences between the VNA titers and seropositivity rates obtained with IPVvac in the two methods tested. AEvac, however, elicited significantly lower titers than those observed in the group receiving inactivated vaccine. In addition, the profiles of antirabies IgG antibodies, evaluated by ELISA, and VNA, appraised by cell-culture neutralization test, were slightly different, when both vaccines were compared.