Molecular characterization of the avian adeno-associated virus and its use for the development of a viral vector system
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The usefulness of viral-vectored delivery of genetic information to cells and tissues, both in vivo and in vitro, has been well documented. Advances in recombinant DNA technologies and cellular biology have made possible the use of recombinant viruses to correct underlying genetic defects and to express immunogenic peptides to induce protective immune responses in animals. The objective of this work is the use of a non-pathogenic, replication defective avian parvovirus, the avian adeno-associated virus (AAAV), to generate a virus-based system for gene delivery in poultry. Aiming at this purpose we have cloned and sequenced the two known strains of the AAAV (VR-865 and DA-1). Complete infective viral particles of both strains were rescued from these clones, using a previously described system that includes the use of the HEK 293 cell line and a plasmid that encode for some of the immediate early genes of the human adenovirus type 5. These infectious clones obtained were used to generate a plasmid-based system for the production of recombinant AAAV particles coding for the LacZ gene as a reporter. Recombinant viral populations obtained from these plasmids were used to infect primary chicken embryo cell cultures and embryonating chicken eggs. Expression of the reporter gene was observed on both systems. The recombinant plasmids obtained were also used to assess the role of the viral inverted terminal repeats and non-structural proteins on the level and duration of transgene expression in vitro. Results showed that both the inverted terminal repeats and expression of the non-structural proteins of the virus significantly increase the level and duration of transgene expression in cell cultures.