RESUMO
Synthetic peptides mimicking protective B- and T-cell epitopes are good candidates for safer, more effective FMD vaccines. Nevertheless, previous studies of immunization with linear peptides showed that they failed to induce solid protection in cattle. Dendrimeric peptides displaying two or four copies of a peptide corresponding to the B-cell epitope VP1 [136-154] of type O FMDV (O/UKG/11/2001) linked through thioether bonds to a single copy of the T-cell epitope 3A [21-35] (termed B2T and B4T, resp.) afforded protection in vaccinated pigs. In this work, we show that dendrimeric peptides B2T and B4T can elicit specific humoral responses in cattle and confer partial protection against the challenge with a heterologous type O virus (O1/Campos/Bra/58). This protective response correlated with the induction of specific T-cells as well as with an anamnestic antibody response upon virus challenge, as shown by the detection of virus-specific antibody-secreting cells (ASC) in lymphoid tissues distal from the inoculation point.
Assuntos
Linfócitos B/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Dendrímeros/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Ativação Linfocitária , Peptídeos/química , Peptídeos/imunologia , Suínos , VacinaçãoRESUMO
Modifications in the GABA pathway are considered to be responsible for motor alterations in animal models for fragile X-associated tremor ataxia syndrome. We analyzed the expression profile in the cerebellum in a transgenic mouse model that over expresses the human FMR1 gene with CGG repeats in the normal range. We used the "GeneChip Mouse Gene 1.0 ST Array" from Affymetrix analyzing 28,853 well-described and -characterized genes. Based on data from the comparative analysis of the expression profile, we detected a significant gradient with a P value <0.1 and changes in expression equal to or greater than 1.5 times compared to the control mouse genes. There were significant changes in the expression of 104 genes, among which 72% had decreased and 28% had increased expression. With the exception of GabarapL2, no changes in expression of genes from the GABA pathway were observed, which may explain the absence of an altered motor phenotype in these mice. These results further support the view that toxic effects in fragile X-associated tremor ataxia syndrome are due to expansion of CGG repeats rather than increased mRNA levels, since in the transgenic mice the FMR1 mRNA levels were increased 20-100 times compared with those of control littermates.
Assuntos
Cerebelo/citologia , Proteína do X Frágil da Deficiência Intelectual/biossíntese , Proteína do X Frágil da Deficiência Intelectual/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Modelos Animais de Doenças , Síndrome do Cromossomo X Frágil/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Transdução de Sinais/genética , TranscriptomaRESUMO
An analysis of the informative content of sequence stretches on the foot-and-mouth disease virus (FMDV) VPI gene was applied to two important viral serotypes: A and O. Several sequence regions were identified to allow the reconstruction of phylogenetic trees equivalent to those derived from the whole VPI gene. The optimal informative regions for sequence windows of 150 to 250 nt were predicted between positions 250 and 550 of the gene. The sequences spanning the 250 nt of the 3' end (positions 400 to 650), extensively used for FMDV phylogenetic analyses, showed a lower informative content. In spite of this, the use of sequences from this region allowed the derivation of phylogenetic trees for type A and type O FMDVs which showed topologies similar to those previously reported for the whole VP1 gene. When the sequences determined for viruses isolated in Argentina, between 1990 and 1993, were included in these analyses, the results obtained revealed features of the circulation of type A and type O viruses in the field, in the months that preceded the eradication of the disease in this country. Type A viruses were closely related to an Argentinean vaccine strain, and defined an independent cluster within this serotype. Among the type O viruses analysed, two groups were distinguished; one was closely related to the South American vaccine strains, while the other was grouped with viruses of the O3 subtype. In addition, a detailed phylogeny for type A FMDV is presented.
Assuntos
Aphthovirus/genética , Capsídeo/genética , Filogenia , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Aphthovirus/classificação , Sequência de Bases , Capsídeo/química , Proteínas do Capsídeo , DNA Complementar/química , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/química , RNA Viral/genética , Sorotipagem , Proteínas Virais/análiseRESUMO
The origin and evolution of the classical swine fever (CSF) epizootic that occurred in Cuba from 1993 to 1997 has been investigated by the analysis of E2 gene sequences from 15 representative viral isolates as well as the vaccine and the challenge strains used in this country. In the phylogenetic tree derived from these sequences, the Cuban isolates were located in a defined cluster within the previously reported genomic subgroup 1.2. This cluster was related, although distinguishable, from the live vaccine used in Cuba since 1965. Two further groups were identified. One of them included the early viruses isolated in the western part of Cuba until 1996 and the strain Margarita, used for vaccine potency tests since 1965. These results are consistent with the strain Margarita being the origin of the western outbreaks. The viruses isolated from 1996 in eastern Cuba defined a related, but independent group. The level of sequence variation observed in this group does not exclude an independent origin for the eastern isolates.
Assuntos
Vírus da Febre Suína Clássica/genética , Peste Suína Clássica/epidemiologia , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Peste Suína Clássica/fisiopatologia , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/classificação , Vírus da Febre Suína Clássica/isolamento & purificação , Cuba/epidemiologia , DNA Viral/química , DNA Viral/genética , Epidemiologia Molecular , Dados de Sequência Molecular , Filogenia , Suínos , Proteínas do Envelope Viral/químicaRESUMO
A simple reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been developed for the specific amplification of DNA after reverse transcription of RNA from the classical swine fever virus (CSFV). A pair of oligonucleotides was selected from an area of high homology in the genome of CSFV strains, but which differed from the corresponding sequences in the genome of bovine viral diarrhea virus (BVDV) strains. Using these primers (CSFV1-CSFV2), a CSFV specific DNA band of 174 bp was amplified from the CSFV RNA extracted from four reference strains and 14 field isolates, as well as from 25 organ extracts and eight buffy coats and serum samples of experimentally infected animals. No amplification was observed with the RNA from four BVDV reference and vaccine strains and seven field isolates. This RT-PCR assay made it possible, in a one-step reaction, to detect CSFV rapidly, sensitively and specifically in cell culture supernatants and in clinical specimens.
Assuntos
Vírus da Febre Suína Clássica/isolamento & purificação , Peste Suína Clássica/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Bovinos , Vírus da Febre Suína Clássica/genética , Técnica Direta de Fluorescência para Anticorpo , Linfonodos/virologia , Tonsila Palatina/virologia , Pestivirus/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Sensibilidade e Especificidade , Baço/virologia , SuínosRESUMO
A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.