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Clonación y expresión de proteínas recombinantes del virus de la hepatitis E en sistemas heterólogos caracterización y aplicaciones

  • Autores: Nereida Jiménez Oya
  • Directores de la Tesis: Juan Carlos Saiz (dir. tes.)
  • Lectura: En la Universidad Autónoma de Madrid ( España ) en 2010
  • Idioma: español
  • Tribunal Calificador de la Tesis: Albert Bosch i Navarro (presid.), José María Almendral del Río (secret.), Eduardo Gómez-Casado (voc.), Francisco Sobrino Castelló (voc.), Margarita Martín Castillo (voc.), Esther Blanco Lavilla (voc.), Fernando de Ory Manchón (voc.)
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  • Resumen
    • Hepatitis E, an enteric transmitted disease caused by hepatitis E virus (HEV), is responsible for large epidemics and sporadic outbreaks in endemic regions with low levels of hygiene and inadequate water supplies. Lately, the number of reported human autochtonous cases has increased in industrialized countries and a widespread distribution of the infection in the European swine population has also been suggested. HEV swine and human strains are genetically very similar which has raised the hypothesis of a zoonotic potential for the virus. This fact has been further confirmed after viral detection on pig¿ s livers sold in local markets in Europe and U.S. and the description of humans cases after consumption of contaminated uncooked pork and deer meet. All these data highlight the need for better surveillance of viral activity in human and animal populations and, consequently, for more accurate specific tools to address it.

      In this work, different recombinant forms of the open reading frame 2 (ORF2) capsid protein of HEV have been expressed in the baculovirus and the vaccinia virus systems. Both proteins, the complete ORF2 and a truncated form of it lacking the first 111 amino acids of the N-terminal, were efficiently produced and further characterized in both expression systems. Microscopy experiments showed that, in mammalians cells infected with the recombinant vaccinia virus generated, the complete form could be observed in the cell surface, the endoplasmic reticulum and the Golgi apparatus, while the truncated form was localized at the endoplasmic reticulum. Both proteins can be N-glycosylated, although the glycosylation pattern observed was different in the two heterologous systems used. The generated proteins were used to set up an ELISA test for detection of specific anti-HEV antibodies. Best results were obtained with the partially purified truncated form of ORF2 obtained upon infection of insect larvae. The developed ELISAs presented a good specificity and sensitivity both with human and porcine sera and were further validated using a commercial kit. Once optimized, the ELISA was used, in combination with HEVRNA detection by RT-PCR, to analyze the seroprevalence of HEV in over 1400 swine serum samples from different farms and regions across Spain. The results showed a widespread of HEV infection in the country, 20,4% of the animals tested were IgG positive and 18% of them had HEV-RNA in blood. Over 80% of the farms analyzed had, at least, one anti-HEV IgG positive animal and in half of them the infection was active, at least one animal was HEV-RNA positive. Analysis of the immunogenic capability of the generated recombinant proteins demonstrated that all of them elicited specific antibodies on all immunized mice. Immunization was passively transferred to the offspring both by intrauterine and lactation routes, as shown by the presence of specific antibodies in newborns of immunized mothers. Immunogenicity of the proteins was further applied to the generation and characterization of murine monoclonal antibodies.

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