RESUMEN
The recombinant baculocvirus containing genome P1-2A-P3 of hepatitis A virus (HAV) was constructed and used for infecting the Sf9 insect cells. It was demonstrated that the deletion of 2BC from HAV polyprotein and the insertion of a new 3C protease cleavage site between P1-2A and P3 did not interfere with the processing of polyprotein or with forming the 70S-procapsids. The identity of the protein contents as well as of morphological and antigen characteristics, obtained in Sf9-cells, to HAV empty capsids, which take shape in the infected mammal cells, proves that it is possible to use them in making the vaccine and diagnostic preparations.
Asunto(s)
Baculoviridae/metabolismo , Cápside/metabolismo , Hepatitis A/metabolismo , Precursores de Proteínas/metabolismo , Animales , Baculoviridae/genética , Baculoviridae/ultraestructura , Cápside/química , Cápside/ultraestructura , Línea Celular , Eliminación de Gen , Genoma Viral , Hepatitis A/genética , Antígenos de la Hepatitis C/análisis , Antígenos de la Hepatitis C/metabolismo , Immunoblotting , Insectos , Microscopía Inmunoelectrónica , Ingeniería de Proteínas , Precursores de Proteínas/análisis , Precursores de Proteínas/química , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Recombinación GenéticaRESUMEN
The cytolytic form of hepatitis E virus reproduction in FRhk-4 cells is described. The cytopathic effect was observed by days 6-7 of the 25th passage in this cell culture. Reverse transcription with polymerase chain reaction were used to study virus reproduction daily for 10 days postinfection. The virus replication starts on day 4 after inoculation. The development of the cytopathic effect depended on the serum concentration in culture medium, whereas the virus replication did not depend on this factor. The replicative form (-RNA) of viral RNA appeared on day 4 postinoculation, as did the new genomic one (+RNA).
Asunto(s)
Virus de la Hepatitis E/fisiología , Replicación Viral , Animales , Línea Celular , Efecto Citopatogénico Viral , Genoma Viral , Virus de la Hepatitis E/genética , Virus de la Hepatitis E/patogenicidad , Macaca mulatta , Reacción en Cadena de la Polimerasa , ARN Viral/genéticaRESUMEN
The morphogenetic pathway of hepatitis A virus (HAV), classified as a member of the enteroviruses within the Picornaviridae, still remains obscure and seems to differ considerably from that of poliovirus, the most studied representative of this genus. In order to elucidate the precursor/product relationship of HAV structural proteins, subviral particles, which represent more than 50% of the viral antigen produced in infected cells, were separated from mature virions and their polypeptide pattern was analyzed by polyacrylamide gel electrophoresis and immunoblotting using monospecific antisera. Whereas mature virions are composed of viral proteins VP1, VP2, and VP3, subviral particles contained VP0 and smaller polypeptides instead of VP2. Comparison of proteins of different strains of HAV showed that VP0 of strain HAS-15 migrated slower than that of strains MBB or GBM. During the course of the infectious cycle, VP0 accumulated and only small portions were converted to VP2 supporting earlier observations that encapsidation of RNA with concomitant cleavage of VP0 is rate-limiting, leaving a large amount of viral antigen in premature particles. Similar to VP0, accumulation of VP1 was observed and two immunologically related precursor proteins, p38 and p36, were found during the course of infection. Immunological characterization of p38 using antisera directed to the N-terminus of VP1 and to synthetic peptides located at the presumptive C- and N-termini of 2A suggests that p38 is VP1 delta 2A carrying 45 N-terminal amino acids of the P2-region.
Asunto(s)
Hepatovirus/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Estructurales Virales/metabolismo , Animales , Línea Celular , Electroforesis en Gel de Poliacrilamida , Hepatovirus/crecimiento & desarrollo , Immunoblotting , Precursores de Proteínas/aislamiento & purificación , Proteínas Estructurales Virales/aislamiento & purificaciónRESUMEN
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
A modified technique of protein staining with silver nitrate was employed in electrophoretic analysis of hepatitis A virus structural proteins. The modifications of the original technique, i.e. thorough washing of the gel, increased formaldehyde concentration and a more intensive lighting of the gel, have elevated the method sensitivity 10-fold permitting the detection of up to 0.1 ng protein. The modification has allowed detection of VP1, VP2, and VP3 structural proteins with molecular masses 33, 29, and 27 kD, respectively, in virus preparations of low concentrations.