RESUMEN
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.
Asunto(s)
Virus Lassa , Marburgvirus , Proteínas de la Nucleocápside , Proteínas Recombinantes , Animales , Reacciones Antígeno-Anticuerpo , Escherichia coli , Humanos , Fiebre de Lassa/inmunología , Virus Lassa/inmunología , Virus Lassa/aislamiento & purificación , Enfermedad del Virus de Marburg/inmunología , Marburgvirus/inmunología , Marburgvirus/aislamiento & purificación , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Suero/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/inmunología , Proteínas del Sistema Complemento/inmunología , Virus Lassa/química , Proteínas Virales/química , Animales , Ensayo de Inmunoadsorción Enzimática , Virus Lassa/inmunología , Ratones , Conformación Proteica , Huella de Proteína , Proteínas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/diagnóstico , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre de Lassa/diagnóstico , Enfermedad del Virus de Marburg/diagnóstico , Pruebas Serológicas/normas , Secuencia de Aminoácidos , Anticuerpos Antivirales/sangre , Reacciones Falso Positivas , Humanos , Datos de Secuencia Molecular , Estándares de Referencia , Alineación de Secuencia , Proteínas Virales/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Infecciones por Arenaviridae/diagnóstico , Arenaviridae/inmunología , Inmunoglobulina G/biosíntesis , Isotipos de Inmunoglobulinas/biosíntesis , Fiebre de Lassa/diagnóstico , Virus Lassa/inmunología , Coriomeningitis Linfocítica/diagnóstico , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Anticuerpos Antivirales/sangre , Afinidad de Anticuerpos , Reacciones Cruzadas , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Inmunización , Inmunoglobulina G/sangre , Isotipos de Inmunoglobulinas/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBARESUMEN
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.
Asunto(s)
Virus Lassa/inmunología , Proteínas Estructurales Virales/inmunología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Cápside/inmunología , Epítopos , Glicoproteínas/inmunología , Sueros Inmunes/análisis , Inmunización/métodos , Técnicas para Inmunoenzimas , Conejos , Proteínas del Núcleo Viral/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Arenavirus del Nuevo Mundo/inmunología , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos Virales/inmunología , Reacciones Cruzadas/inmunología , Epítopos/inmunología , Femenino , Hibridomas/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Células Vero , Cultivo de VirusRESUMEN
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.
Asunto(s)
Orthohantavirus/análisis , ARN Viral/análisis , Proteínas Virales/análisis , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Ensayo de RadioinmunoprecipitaciónRESUMEN
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.
Asunto(s)
Arenaviridae/genética , Poli A/genética , ARN Mensajero/genética , Moldes Genéticos , Animales , Arenavirus del Nuevo Mundo/genética , Chlorocebus aethiops , Virus Lassa/genética , ARN Viral/genética , Células VeroRESUMEN
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.
Asunto(s)
Arenaviridae/genética , Cápside/genética , Virus Lassa/genética , ARN Mensajero/aislamiento & purificación , ARN Viral/aislamiento & purificación , Proteínas del Núcleo Viral/genética , Animales , Fiebre de Lassa/microbiología , Poli A/genética , Poli A/aislamiento & purificación , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Células VeroRESUMEN
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.
Asunto(s)
Arenaviridae/análisis , Cápside/análisis , ARN Mensajero/análisis , ARN Viral/análisis , Proteínas del Núcleo Viral/análisis , Animales , Arenaviridae/genética , Arenaviridae/patogenicidad , Arenavirus del Nuevo Mundo/análisis , Arenavirus del Nuevo Mundo/genética , Arenavirus del Nuevo Mundo/patogenicidad , Cápside/genética , Electroforesis en Gel de Poliacrilamida/métodos , Virus Lassa/análisis , Virus Lassa/genética , Virus Lassa/patogenicidad , Pruebas de Precipitina/métodos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Conejos , Proteínas del Núcleo Viral/genética , Cultivo de VirusRESUMEN
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.
Asunto(s)
Arenaviridae/genética , Arenavirus del Nuevo Mundo/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Animales , Arenavirus del Nuevo Mundo/metabolismo , Cápside/biosíntesis , Cápside/genética , Células Vero , Proteínas del Núcleo Viral/biosíntesis , Proteínas del Núcleo Viral/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
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.