RESUMO
Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.
Assuntos
Vírus Lassa , Marburgvirus , Proteínas do Nucleocapsídeo , Proteínas Recombinantes , Animais , Reações Antígeno-Anticorpo , Escherichia coli , Humanos , Febre Lassa/imunologia , Vírus Lassa/imunologia , Vírus Lassa/isolamento & purificação , Doença do Vírus de Marburg/imunologia , Marburgvirus/imunologia , Marburgvirus/isolamento & purificação , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/imunologia , Proteínas do Nucleocapsídeo/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Soro/imunologiaRESUMO
Addition of the complement to the antigen in Lassa virus ELISA stimulated the sensitivity and specificity of the assay. The effect depended on the mouse Ig subclasses. ELISA plate sensitization with IgG2a and IgG2b increased the sensitivity of ELISA 8-16 times. Protein footprinting data suggest that the complement C1q component stimulates antibody-induced conformation changes of the antigen.
Assuntos
Anticorpos Antivirais/imunologia , Proteínas do Sistema Complemento/imunologia , Vírus Lassa/química , Proteínas Virais/química , Animais , Ensaio de Imunoadsorção Enzimática , Vírus Lassa/imunologia , Camundongos , Conformação Proteica , Pegadas de Proteínas , Proteínas Virais/imunologiaRESUMO
Sera of normal subjects and AIDS patients living in Minsk and Odessa were tested for antibodies to hazardous viral infections Lassa, Marburg, and Ebola. Four to 16% of examinees were seropositive to Ebola virus, 0.8 to 2.3% to Lassa, and up to 0.8% to Marburg virus. Common B-epitopes were found in viruses belonging to different families: Lassa, Ebola, and HIV. Antibodies specific to these viruses antigens were found in the reference sera to influenza A and B, respiratory syncytial virus, and adenovirus. Sera of convalescents after malaria and of AIDS patients contained antibodies to Lassa virus.
Assuntos
Síndrome da Imunodeficiência Adquirida/diagnóstico , Doença pelo Vírus Ebola/diagnóstico , Febre Lassa/diagnóstico , Doença do Vírus de Marburg/diagnóstico , Testes Sorológicos/normas , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Reações Falso-Positivas , Humanos , Dados de Sequência Molecular , Padrões de Referência , Alinhamento de Sequência , Proteínas Virais/químicaRESUMO
The dynamics of the induction of individual IgG subclasses in BALB/c and CBA mice and their role on the diagnostics of arenaviruses LCM, Lassa and Mopeia was studied. The study demonstrated that in solid-phase enzyme immunoassay (EIA) isotypes IgG2a and IgG2b to virus Mopeia could be used for the differentiation of virus Mopeia from viruses LCM and Lassa, and the antigen of virus Mopeia could be used for the preparation of diagnostic EIA systems not only to nonpathogenic virus Mopeia, but also to pathogenic viruses LCM and Lassa.
Assuntos
Anticorpos Antivirais/biossíntese , Infecções por Arenaviridae/diagnóstico , Arenaviridae/imunologia , Imunoglobulina G/biossíntese , Isotipos de Imunoglobulinas/biossíntese , Febre Lassa/diagnóstico , Vírus Lassa/imunologia , Coriomeningite Linfocítica/diagnóstico , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Anticorpos Antivirais/sangue , Afinidade de Anticorpos , Reações Cruzadas , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática , Imunização , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBARESUMO
The immunogenic properties of Lassa virus GP1, GP2, and NP polypeptides were studied in rabbits. Lassa virus NP polypeptide, in contrast to GP1 and GP2 polypeptides, was shown to induce the highest titres of antibodies determined by IFA and ELISA tests. Moreover, the antibody relative avidity experiment showed that the anti-NP antibodies has ELISA index of 0.63 whereas anti-GP1 and anti-GP2 antisera had those of 0.49 and 0.28, respectively.
Assuntos
Vírus Lassa/imunologia , Proteínas Estruturais Virais/imunologia , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Capsídeo/imunologia , Epitopos , Glicoproteínas/imunologia , Soros Imunes/análise , Imunização/métodos , Técnicas Imunoenzimáticas , Coelhos , Proteínas do Core Viral/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
Six monoclonal antibody-producing hybridoma cell lines were generated by fusion of NS-1 myeloma cells with BALB/c immune splenocytes. Monoclonal antibodies (MCA) specific to Machupo virus NP protein were used to study cross-reactivity between pathogenic and nonpathogenic arenaviruses. It was shown that 3140 MCA cross-reacted in IFA with Lassa, Tacaribe, and Tamiami arenaviruses whereas 3101 MCA reacted with Machupo virus alone. It was assumed that these 3101 MCA could be used for differentiation of Machupo virus in IFA.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Arenavirus do Novo Mundo/imunologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Antígenos Virais/imunologia , Reações Cruzadas/imunologia , Epitopos/imunologia , Feminino , Hibridomas/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Células Vero , Cultura de VírusRESUMO
Virus-specific proteins G1, G2, and N with molecular weights of 70, 55-57, and 50 kilodaltons, respectively, were detected by radioimmunodiffusion tests in VERO E-6 cells infected with strains of virus of hemorrhagic fever with renal syndrome (HFRS) isolated in the European USSR from a patient with HFRS, a fatal human case of HFRS (the strains K-27 and P-360) and from a bank vole (strain CG-1820). The sera from human convalescents after HFRS in the European USSR and rat sera prepared with the CG-1820 strain precipitated proteins possessing similar electrophoretic characteristics from a lysate of cells infected with the CG-1820, K-27 and P-360 strains. The sera from human HFRS convalescents in the Far East did not precipitate protein Gl. The viral RNA derived by immunosorption method from intracellular nucleocapsids of CG-1820 strain and strain 4590 (isolated from Ap. Peninsulae in the Far East) contained 3 classes of molecules: L, M, and S. L- and M-RNA of these strains had the same molecular weight (1.62 and 1.38 megadaltons). The molecular weight of S-RNA of the strain 4590 was 0.76 megadalton and that of the CG-1820 strain 0.83 megadalton. It is assumed that there are species differences among the viruses, causative agents of HFRS, circulating in the European and Far East regions of the USSR.
Assuntos
Orthohantavírus/análise , RNA Viral/análise , Proteínas Virais/análise , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Ensaio de RadioimunoprecipitaçãoRESUMO
The total RNA from cells infected with Machupo and Lassa viruses as well as poly(A+) and poly(A-) fractions of the RNA were translated in the cell-free protein synthesizing system from rabbit reticulocytes. The translated products were treated with specific antibodies and analyzed in polyacrylamide gel electrophoresis. Only poly(A-) fraction of RNA coded for the synthesis of NP protein in vitro. The mRNAs for NP protein of Machupo and Lassa viruses are supposed to contain no poly(A) sequences at 3'end, or if they really do, the size of the sequences is not adequate for binding with oligo(dT)-cellulose.
Assuntos
Arenaviridae/genética , Poli A/genética , RNA Mensageiro/genética , Moldes Genéticos , Animais , Arenavirus do Novo Mundo/genética , Chlorocebus aethiops , Vírus Lassa/genética , RNA Viral/genética , Células VeroRESUMO
Single-stranded RNAs from Lassa virus-infected cells were fractionated by affinity chromatography on an oligo(dT)-cellulose column as well as by sucrose gradient centrifugation. Fractionated RNAs were translated in rabbit reticulocyte lysates, and translation products were precipitated with monoclonal antibodies to the nucleocapsid protein of Lassa virus. It has been shown that mRNA for the nucleocapsid protein is 15-16S and the RNA does not contain long if any poly(A) sequences.
Assuntos
Arenaviridae/genética , Capsídeo/genética , Vírus Lassa/genética , RNA Mensageiro/isolamento & purificação , RNA Viral/isolamento & purificação , Proteínas do Core Viral/genética , Animais , Febre Lassa/microbiologia , Poli A/genética , Poli A/isolamento & purificação , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , Células VeroRESUMO
Total RNA from cells infected with Machupo and Lassa viruses as well as individual sedimentation classes of these RNAs were translated in the cell-free protein-synthesizing system from rabbit reticulocytes. The translation products were precipitated either with anti-Machupo immune gamma-globulin or monoclonal antibodies to nucleocapsid protein (NP) of Lassa virus. Both total RNA and RNA fraction with the sedimentation coefficient 15-16S promoted the synthesis of protein which comigrated in gel with NP protein of purified virions. It is concluded that monocistron mRNA for NP protein of arenaviruses has the sedimentation coefficient 15-16 S.
Assuntos
Arenaviridae/análise , Capsídeo/análise , RNA Mensageiro/análise , RNA Viral/análise , Proteínas do Core Viral/análise , Animais , Arenaviridae/genética , Arenaviridae/patogenicidade , Arenavirus do Novo Mundo/análise , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/patogenicidade , Capsídeo/genética , Eletroforese em Gel de Poliacrilamida/métodos , Vírus Lassa/análise , Vírus Lassa/genética , Vírus Lassa/patogenicidade , Testes de Precipitina/métodos , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , Coelhos , Proteínas do Core Viral/genética , Cultura de VírusRESUMO
RNA from Machupo virus infected cells was centrifuged in a linear sucrose gradient and RNAs from gradient fractions were tested separately for template activity in a cell-free protein synthesizing system from rabbit reticulocytes. Fraction 15-16 S programmed the synthesis of protein that migrated in SDS-polyacrylamide gel like the nucleocapsid protein of a purified virus. The synthesis of virus glycoproteins was not detected in the system.
Assuntos
Arenaviridae/genética , Arenavirus do Novo Mundo/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Viral/genética , Animais , Arenavirus do Novo Mundo/metabolismo , Capsídeo/biossíntese , Capsídeo/genética , Células Vero , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/genéticaRESUMO
The effect of inhibitors of mRNA synthesis (actinomycin D and alpha-amanitin) and DNA replication (mitomycin C and ethidium bromide) on Machupo virus reproduction was studied. Actinomycin D (1.0-4.0 micrograms/ml) and alpha-amanitin (10.0 micrograms/ml) added immediately after adsorption reduced the infectious virus titre by 2 lg or more. Actinomycin D inhibited virus reproduction not only at the early but also at the later stages of infection. Mitomycin C and ethidium bromide did not inhibit Machupo virus reproduction. The cells treated with actinomycin D at early and late stages of infection were found to contain immunoprecipitable virus-specific proteins 78, 64, and 37 KD described previously. The total amount of virus-specific proteins in the inhibitor-treated cells was reduced 3.7 and 2,6-fold after addition of actinomycin at 0 and 12 h postinfection, respectively, as compared with untreated cells. The mechanism of action of actinomycin D on the Machupo virus reproduction is discussed.
