RESUMEN
The hepatitis A virus (HAV) capsid protein VP1, VP2 and VP3 are exposed at the virion surface and should therefore contain antigenic determinants. Algorithms for hydrophilicity, antigenicity and flexibility were used to predict probable antigenic sites. Synthesis of 7- to 23-membered overlapping peptides from seven sites, viz., 1-11, 1-17, 2-33, 11-25, 73-82, 76-86, 98-109, 98-112, 102-107, 102-108, 108-127, 113-123, 118-140, 276-298 from VP1, 42-62 from VP2, 76-85 from VP3, and 1-23 from VP4, was performed by various solid-phase methods. Free peptides and their conjugates with different carriers were used for immunization and study of antigenicity. The peptides did not interact with antibodies to the hepatitis A virus, whereas their conjugates did not induce the formation of anti-HAV-antibodies.
Asunto(s)
Cápside/metabolismo , Hepatovirus/metabolismo , Péptidos/síntesis química , Secuencia de Aminoácidos , Antígenos Virales/inmunología , Epítopos/inmunología , Hepatovirus/inmunología , Inmunohistoquímica , Datos de Secuencia Molecular , Péptidos/metabolismoRESUMEN
A modified enzyme immunoassay based on adsorption of antihepatitis A virus (HAV) IgG-HRPO conjugate and monoclonal antibodies to HAV were used to investigate antigenic differences between mature HAV virions and subviral particles with different buoyant densities in CsCl produced in HAV-infected cells. The mature virions (1.34 g/cm3) appeared to have common antigenic determinants with subviral particles (1.20, 1.27, and 1.30 g/cm3) and possess some additional determinants. Nevertheless, both subviral particles and mature virions induced antibodies capable of neutralizing HAV infectivity in tissue culture.
Asunto(s)
Antígenos Virales/inmunología , Hepatovirus/inmunología , Virión/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/sangre , Fenómenos Químicos , Química Física , Cobayas , Inmunización , Técnicas para Inmunoenzimas , Pruebas de Neutralización , ConejosRESUMEN
Peptide (P6) representing amino acids 62-75 of HAV capsid protein VP3 was synthesized. Pure P6, octamer form of P6, and P6 conjugated to KLH were injected into rabbits. The sera against these preparations reacted with denaturized VP3 in immunoblotting tests; however, only anti-P6-KLH serum was capable of binding the native HAV.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Sitios de Unión de Anticuerpos/efectos de los fármacos , Hepatovirus/inmunología , Fragmentos de Péptidos/inmunología , Péptidos/inmunología , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Electroforesis en Gel de Poliacrilamida , Sueros Inmunes/aislamiento & purificación , Inmunización/métodos , Immunoblotting , Epítopos Inmunodominantes/inmunología , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Péptidos/síntesis química , Péptidos/genética , Conejos , Proteínas Virales/síntesis química , Proteínas Virales/genéticaRESUMEN
The Western blot procedure with highly specific antipeptide antibody was applied to identify the electrophoretic mobility of hepatitis A virus capsid proteins. Polypeptides with molecular weights of 33, 29 and 27 kDa proved to be VP1, VP2, and VP3 proteins as they reacted with sera generated to VP1 recombinant protein and to synthetic oligopeptides 42-62 VP2 and 62-75 VP3, respectively.
Asunto(s)
Hepatovirus/análisis , Mapeo Peptídico/métodos , Proteínas Estructurales Virales/análisis , Animales , Anticuerpos Antivirales/sangre , Western Blotting , Electroforesis en Gel de Poliacrilamida , Cobayas , Hepatovirus/inmunología , Humanos , Sueros Inmunes , Inmunización , Técnicas para Inmunoenzimas , Peso Molecular , Conejos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Proteínas Estructurales Virales/inmunologíaRESUMEN
Hepatitis A virus (HAV) was propagated in continuous rhesus monkey embryo kidney cells (FRhK-4 line). HAV RNA extracted with phenol from purified HAV pretreated with proteinase K was used for molecular cloning experiments. Two radioactive plasmids, pHAV-23 and pHAV-5p, containing HAV cDNA fragments complementary to structural and nonstructural parts of HAV genome, respectively, were found to be useful for discrimination between mature virions and subviral structures. HAV cDNA-RNA hybridization seems to be 10 times more sensitive than the enzyme immunoassay (ELISA).
Asunto(s)
Hepatovirus/genética , Hibridación de Ácido Nucleico , ARN Viral/análisis , Animales , Clonación Molecular/métodos , ADN/genética , ADN Viral/genética , Heces/microbiología , Hepatitis A/diagnóstico , Humanos , Técnicas para Inmunoenzimas , Macaca mulatta , Enfermedades de los Monos/diagnóstico , Radioisótopos de Fósforo , Sondas ARN , ARN Viral/genética , ARN Viral/aislamiento & purificación , Cultivo de VirusRESUMEN
Four types of virus-specific particles with different sedimentation coefficients and buoyant densities in CsCl were shown to be accumulated in hepatitis A virus (strain HAS-15) infected fetal rhesus monkey kidney cells (FRhK-4 line). Unlike the mature virions (155S, 1.34 g/cm3), cell-associated isosedimenting 92 S-particles (buoyant densities of 1.30 and 1.20 g/cm3) proved to be sensitive to lipase action. Particles of all four types were shown to contain similar sets of polypeptides, and, with the exception of "empty" 1.30 g/cm3-particles, appeared to be "full" under the immune electron microscopic examination. The viral RNA was unequivocally identified by the molecular hydridization test only in the mature virions.
Asunto(s)
Hepatovirus/análisis , Virión/análisis , Animales , Antígenos Virales/análisis , Línea Celular , Centrifugación por Gradiente de Densidad , Hepatovirus/inmunología , Hepatovirus/fisiología , Macaca mulatta , Péptidos/análisis , Proteínas Virales/análisis , Virión/inmunología , Virión/fisiología , Replicación ViralRESUMEN
The propagation time-course of hepatitis A virus (HAV, strain HAS-15) in continuous culture of the foetal rhesus monkey kidney cells (FRhK-4) was investigated. The HAV infectivity and viral RNA content in the infected cells reached the maximal level 5-8 days after infection, while accumulation of hepatitis A antigen (HAAg) continued for 2-3 weeks more. Viral particles with the densities 1.27-1.28 g/cm3 and 1.18-1.22 g/cm3 were isolated from the infected cells as well as the mature virions with the buoyant density 1.33-1.34 g/cm3 in CsCl. The concurrent accumulation of mature virus and "light" particles (1.18-1.22 g/cm3) was registered during infection. Viral particles with the density 1.27-1.28 g/cm3 accumulated predominantly from the 14th to the 21st-24th days after infection. The mature virions (1.34 g/cm3) as well as the particles with the density 1.24-1.25 g/cm3 were isolated from supernatant precipitated by ammonium sulphate. The HAAg activity of both fractions increased progressively in equal proportion in course of infection.
Asunto(s)
Hepatovirus/fisiología , Virión/fisiología , Replicación Viral , Animales , Antígenos Virales/análisis , Línea Celular , Fenómenos Químicos , Química Física , Antígenos de Hepatitis A , Hepatovirus/análisis , Hepatovirus/inmunología , Macaca mulatta , Virión/análisis , Virión/inmunología , Cultivo de VirusRESUMEN
"Light" viral antigen (HAAg) with buoyant density 1.20 g/cm2 and sedimentation coefficient 92S are accumulated together with mature viral particles in Hepatitis A virus (HAV) infected FRhK-4 cells. This HAAg is localized predominantly in endoplasmic reticulum fraction of infected cells, while nature virions are localized in cytosol. In contrast to mature virus, "light" HAAg is sensitive to trypsin digestion and is not able to hybridize with synthetic oligodeoxinucleotide which is complementary to structural part of HAV RNA. Antigenic properties of mature virus and "light" antigen are compared.
Asunto(s)
Hepatovirus/patogenicidad , Animales , Antígenos Virales/análisis , Fenómenos Biofísicos , Biofisica , Membrana Celular/microbiología , Hepatitis A/inmunología , Hepatitis A/microbiología , Hepatovirus/inmunología , Hepatovirus/fisiología , Péptidos/análisis , ARN Viral/análisis , Virión/inmunología , Virión/patogenicidad , Virión/fisiología , Cultivo de Virus , Replicación ViralRESUMEN
The possibility of combined performance of radioimmunoassay (RIA) and immune electron microscopy (IEM) in one preparation using protein A of Staphylococcus aureus for hepatitis A virus (HAV) detection in fecal specimens from hepatitis A patients within a short time (40-50 min) has been demonstrated. In the examinations of one preparation by RIA and IEM, their sensitivity was found to be approximately similar. According to RIA, a high content of HAV antigen was observed in those preparations where in addition to typical particles there were structures resembling individual fragments of empty HAV particles coated with antibody.
Asunto(s)
Hepatovirus/aislamiento & purificación , Antígenos Virales/análisis , Heces/microbiología , Hepatovirus/inmunología , Hepatovirus/ultraestructura , Humanos , Microscopía Electrónica/métodos , Radioinmunoensayo/métodos , Proteína Estafilocócica A , Factores de Tiempo , Virión/aislamiento & purificaciónRESUMEN
The effect of preliminary administration of alpha-tocopherol acetate (1 mg/kg) and sodium selenite (1 mg/kg) on RNA synthesis rate and changes in the macroergic content in ischemic myocardium was studied. It was shown to be conducive to ATP level maintenance after 30- and 60-minute heart ischemia. A statistically significant accumulation of RNA by the myocardium following I-hour ischemia as well as this polynucleotide synthesis activation were recorded. Preservation of the energetic resources by the cell along with RNA metabolism returning to normal contributes to inhibiting the emergence of irreversible cell damage and increases regenerative potentialities of the myocardium.