Assuntos
DNA/isolamento & purificação , Contaminação de Medicamentos , Vacina Antipólio Oral/isolamento & purificação , Animais , Linhagem Celular , Células Cultivadas , DNA/análise , Eletroforese em Gel de Ágar , Immunoblotting , Filtros Microporos , Peso Molecular , Hibridização de Ácido Nucleico , Vacina Antipólio Oral/análise , EspectrofotometriaRESUMO
According to the WHO requirements, the concentration of cellular DNA in vaccine preparations produced by pooling virus from continuous cell lines is limited to 100 ng/dose. In this study, different methods were used for purification of tick-borne encephalitis virus suspensions grown in continuous cultures of cell line 4647 from cellular DNA. Two approaches are proposed based on treatment with DNAse and promamin sulfate which allow one to reduce cellular DNA concentration in the virus preparation to the acceptable level. Prospects of their use in vaccine production are discussed.
Assuntos
DNA/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Animais , Centrifugação com Gradiente de Concentração/métodos , Centrifugação Isopícnica/métodos , Cromatografia DEAE-Celulose/métodos , Cromatografia em Gel/métodos , DNA/análise , Desoxirribonuclease I/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Filtros Microporos , Protaminas/farmacologia , Vacinas de Produtos Inativados/análise , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Vacinas Virais/análise , Vacinas Virais/imunologia , Vacinas Virais/isolamento & purificaçãoRESUMO
Initiation of poliovirus RNA translation in reticulocyte lysates is mainly not precise, i.e. it occurs at the sites in the middle of the viral genome but not at the beginning of the polyprotein reading frame. The anomaly is due to the deficiency of translation initiation factors. Partial purification of the protein fraction stimulating the precise translation from the Krebs-2 cells is reported in the paper. This fraction, like the crude lysates factors, was considerably less active with the RNA of attenuated poliovirus strains of type 1 and 3 than with the RNA of virulent strains. The change in interaction of the specific segment of viral RNA with the translation initiation factors is suggested to contribute to the attenuated phenotype of the Sabin poliovirus strains.
Assuntos
Regulação da Expressão Gênica , Genes Virais , Mutação , Fatores de Iniciação de Peptídeos/genética , Poliovirus/genética , Poliovirus/imunologia , Vacina Antipólio OralRESUMO
Conditions that permitted cell-free synthesis of at least one of the non-structural, in addition to two-structural, polypeptides of tick-borne encephalitis virus have been found. The time course of accumulation of virus-specific polypeptides in extracts of Krebs-2 cells and reticulocyte lysates as well as the peptide maps of the products synthesised were studied. A model of generation of viral structural polypeptides has been proposed, according to which a common precursor of these proteins while in a nascent form, is processed in a membrane-dependent reaction into a C-terminal segment, corresponding to the polypeptide moiety of envelope glycoprotein E, and an N-terminal segment, doublet p36/33. Subsequently, an N-terminal segment, corresponding to the core polypeptide C, is cleaved off from p36/33. The remaining C-terminal segment of p36/33 is possibly a precursor of the membrane polypeptide M. The translational strategy of flaviviruses is compared to that of other positive-stranded RNA viruses.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Biossíntese de Proteínas , RNA Viral/genética , Proteínas Virais/biossíntese , Animais , Linhagem Celular , Sistema Livre de Células , Vírus da Encefalite Transmitidos por Carrapatos/metabolismo , Cinética , Biossíntese Peptídica , Coelhos , Reticulócitos/metabolismo , Reticulócitos/microbiologia , Proteínas Virais/genética , Proteínas Estruturais ViraisRESUMO
The nucleotide sequence of the RNase T1-resistant fragment of encephalomyocarditis virus RNA that includes the poly(C) tract was determined by gel sequencing and mobility shift methods. This sequence is (5') AC126(127) UCUCUCUC9UAACG (3'). The results show that the poly(C) tract is discontinuous, i.e., it is interrupted by the UCUCUCU-sequence. The tract displays anomalously high mobility in polyacrylamide gels as compared to random polynucleotides, indicating that electrophoretic determination of its length gives underestimated values.
