RESUMO
Classical swine fever virus (CSFV) causes severe disease in pigs associated with leukopenia, haemorrhage and fever. We show that CSFV infection protects endothelial cells from apoptosis induced by the dsRNA mimic, pIpC, but not from other apoptotic stimuli, FasL or staurosporine. CSFV infection inhibits pIpC-induced caspase activation, mitochondrial membrane potential loss and cytochrome c release as well as the pro-apoptotic effects of truncated Bid (tBid) overexpression. The CSFV proteins N(pro) and E(rns) both contribute to CSFV inhibition of apoptosis. We conclude that CSFV infection can inhibit apoptotic signalling at multiple levels, including at the caspase-8 and the mitochondrial checkpoints. By supporting viral replication, endothelial cells may promote CSFV pathogenesis.
Assuntos
Apoptose/efeitos dos fármacos , Vírus da Febre Suína Clássica/patogenicidade , Células Endoteliais/fisiologia , RNA de Cadeia Dupla/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Aorta/virologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/virologia , Ativação Enzimática , Suínos , Proteínas do Envelope Viral/fisiologiaRESUMO
Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.
Assuntos
Vírus da Febre Suína Clássica/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Animais , Células Cultivadas , Peste Suína Clássica/diagnóstico , Vírus da Febre Suína Clássica/genética , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/isolamento & purificação , Reações Falso-Positivas , RNA Viral/análise , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , SuínosRESUMO
Mutations in the BANYULS (BAN) gene lead to precocious accumulation of anthocyanins in immature seed coat in Arabidopsis. The ban -1 allele has been isolated from a collection of T-DNA transformants and found to be tagged by the integrative molecule. The sequencing of wild-type and two independent mutant alleles confirmed the identity of the gene. Analysis of the full-length cDNA sequence revealed an open reading frame encoding a 342 amino acid protein which shared strong similarities with DFR and other enzymes of the phenylpropanoid biosynthesis pathway. BAN expression was restricted to the endothelium of immature seeds at the pre-globular to early globular stages of development as predicted from the maternal inheritance of the phenotype, and therefore represents a marker for early differentiation and development of the seed coat. BAN is probably involved in a metabolic channelling between the production of anthocyanins and pro-anthocyanidins in the seed coat.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/enzimologia , Arabidopsis/genética , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Sementes/enzimologia , Sementes/crescimento & desenvolvimento , Alelos , Sequência de Aminoácidos , Antocianinas/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/embriologia , Catequina/análise , Clonagem Molecular , Herança Extracromossômica , Flavonoides/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação/genética , NADH NADPH Oxirredutases/química , Filogenia , Estruturas Vegetais/enzimologia , Estruturas Vegetais/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Sementes/anatomia & histologia , Sementes/genética , Alinhamento de SequênciaRESUMO
An assay was developed in which reverse transcription (RT), nested polymerase chain reaction (PCR) and accumulation of amplicon-specific fluorescence could take place in a single, closed reaction tube. The assay, which was classical swine fever virus RNA-specific, was compared with other methods for detection of this virus, including various RT-PCR configurations, virus isolation and ELISA. The new method was very sensitive, and less prone to giving false positive results compared to nested PCR carried out in separate reaction tubes. Substitution of different fluorescent probes resulted in specific tests for border disease virus and for bovine viral diarrhoea type II (BVD-II), and one that could detect all pestiviruses except for some BVD-II viruses.
