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.

Immunopathology of rabies infection in mice selected for high or low acute inflammatory reaction

Rabies is a severe and lethal disease that produces a slight inflammatory response during the infection process. We analyzed the immunopathological mechanisms that occur in the central nervous system (CNS) using mice genetically selected for maximal or minimal acute inflammatory reaction (AIRmax or AIRmin). As viral samples, we adopted the antigenic variant 3 (AgV3) of rabies virus from hematophagous bats and a fixed virus strain (PV1 43/3). Titration of specific antibodies was performed using enzyme-linked immunosorbent assay (ELISA). We observed a slight increase in IgG and IgG1 isotypes in infected AIRmax mice. Incubation period, determined by intracerebral inoculation with 100 LD50, was 6-7 days for PV1 43/4 strain and 9-10 days for AgV3. No difference in viral replication was noticed between AIRmax and AIRmin mice. Mortality was 100 percent with both viral strains. Histopathological analysis of brains and spinal cords showed inflammatory foci in all regions of the CNS. No differences were noticed in the number of neutrophils. Negri bodies were observed in practically all sites analyzed. Results suggested that inflammatory reaction is not a determining factor in the susceptibility to rabies infection.

Lack of correlation between rabies virus replication in the brain and antibody isotype profile in genetically modified mice

The relationship among the phenotypes resistance to infection, virus replication in the brain and isotype production was investigated in genetically modified High (H) or Low (L) antibody responder mouse lines. Although they express the same innate susceptibility to rabies infection, these lines differ as to different viral replication rates in the central nervous system and L mice showed a higher permissible state. After intramuscular infection with the Pasteur rabies strain (PV), the H-L interline differences on the earlier stage of virus replication were 1000 and 80 folds on days 5 and 6, respectively. The isotype profile in sera of the experimentally infected mice reflected an interline difference of 25 folds for IgG2a throughout the infection period, and for the IgE production the H-L difference was highly significant only at the beginning of the process. These results confirm the multi-specific effect of antibody immune responsiveness and the general isotype distribution of antibodies in these genetically selected mice. Contrary to the clear correlation between antibody responsiveness and the acquired resistance to rabies infection, the present study demonstrates that the constitutive genetic character of High and Low responder individuals does not intervene in the degree of resistance following infection. Altogether, this study contributes to the knowledge of the protective role of the general innate responsiveness on the pathological pattern to rabies virus infection

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.

Inactivated suckling mouse brain rabies vaccine provides short-term immunity in 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, 60, 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 IU/ml after vaccination, but the immune response persisted for only 122.3 +/- 32.6 days. The SMBV was able to induce immune response in the capuchin monkeys, but protection was short-lived.

Vírus rábico isolado de morcego frugívoro (Artibeus lituratus), capturado em 1997 no município de Rio Claro, SP / Rabies virus isolated from a frugivorous bat (Artibeus lituratus), capturated in 1997 in Rio Claro, Säo Paulo, SP

O vírus rábico foi isolado de morcego frugívoro Artibeus lituratus, capturado no município de Rio Claro, SP, em bairro residencial, em 1997. Neste município, o último caso de raiva animal ocorreu em 1986, sendo este o primeiro relato do isolamento em morcego frugívoro. As implicaçöes em Saúde Pública foram discutidas

[Isolation of rabies virus from an insectivorous bat, Nyctinomops macrotis, in southeast Brazil]. / Isolamento do vírus rábico em morcego insetívoro, Nyctinomops macrotis, no Município de Diadema, SP (Brasil).

The rabies virus was isolated from an insectivorous bat, Nyctinomops macrotis, trapped in Diadema, SP, Brazil, in a public building near a water supply reservoir. Fluorescent antibodies against rabies virus were detected in cerebral tissue and the viral isolation was made after the inoculation of cerebral tissue and salivary gland suspension in mice. There have been no recorded cases of animal rabies in Diadema since 1982, and this is the first isolation of the rabies virus in an insectivorous bat in the city.

Immunomodulatory effect of glucan on the response to experimental antirabies vaccination.

The objective of the present study was to determine the stimulatory response to antirabies vaccination promoted by glucan in mice. Glucan increased both resistance to infection and antibody titres and this effect was more evident when glucan was used at dose of 0.5 mg, administered intraperitoneally before, during and after immunization and when the challenge virus was applied to the foot-pad.

Reduced schedule of human anti-rabies immunization with Fuenzalida & Palacios vaccine. Additional data

It was reevaluated a reduced schedule for anti-rabies post-exposure immunization with newborn mice nervous tissue vaccine (Fuenzalida & Palacios) in a group of 30 non exposed volunteers. The vaccine was administered by intramuscular injections on days zero, 2, 4, 16 and 27, in the deltoid area. Antibody levels were determinated by a simplified serum neutralization microtest on days zero, 16 and 37. On days 16 and 37 the antibody levels of the whole group was > or = 0.5 IU/ml and > or = 1.0 IU/ml, respectively. The cell mediated immunity was precociously detected (on day 4) by the delayed type hypersensitivity skin test. Our results show that this reduced schedule elicited an early and effective humoral and cellular immune response. However it is necessary other studies with larger groups of vaccinees in order to obtain definitive conclusion.

Reduced schedule of human anti-rabies immunization with Fuenzalida & Palacios vaccine. Additional data.

It was reevaluated a reduced schedule for anti-rabies post-exposure immunization with newborn mice nervous tissue vaccine (Fuenzalida & Palacios) in a group of 30 non exposed volunteers. The vaccine was administered by intramuscular injections on days zero, 2, 4, 16 and 27, in the deltoid area. Antibody levels were determinated by a simplified serum neutralization microtest on days zero, 16 and 37. On days 16 and 37 the antibody levels of the whole group was > or = 0.5 IU/ml and > or = 1.0 IU/ml, respectively. The cell mediated immunity was precociously detected (on day 4) by the delayed type hypersensitivity skin test. Our results show that this reduced schedule elicited an early and effective humoral and cellular immune response. However it is necessary other studies with larger groups of vaccinees in order to obtain definitive conclusion.

Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies

A simplified fluorescence inhibition microtest (SFIMT) was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT) and the fluorescence inhibition microtest (FIMT). The simplified test showed reproducibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL) gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO). The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.

Simplified fluorescent inhibition microtest for the titration of rabies neutralizing antibodies.

A simplified fluorescence inhibition microtest (SFIMT) was standardized for the evaluation of antirabies serum neutralizing antibodies based on the rapid fluorescent focus inhibition test (RFFIT) and the fluorescence inhibition microtest (FIMT). The simplified test showed reproducibility similar to that of the FIMT with advantages as easier executation and quicker reading. A simple pre-treatment of Brazilian microplates produced for immune enzymatic assays (PROSIL) gave equivalent results and substantial coast reduction, in relation to imported plates (DIFCO). The simplified test can be easily implemented in less sophisticated laboratories, as alternative to the mouse serum neutralization test, still the most largely employed in Brazil, or even to others as RFFIT and FIMT.