Sequence analysis, viral rescue from infectious clones and generation of recombinant virions of the avian adeno-associated virus.
Aiming at the generation of a viral-vectored system for gene delivery and vaccination in poultry, the entire genomes of the VR-865 and DA-1 strains of the avian adeno-associated virus have been cloned and sequenced. Sequence analysis of the clones showed that the genomic distribution of the structural and non-structural protein-coding genes of these viruses is conserved and in agreement with what has been previously described for the primate adeno-associated viruses. Amino acid differences between the avian adeno-associated viruses and the primate adeno-associated viruses are more evident in the genes that code for the non-structural (Rep) proteins of the virus, while the Cap region amino acid sequence was found to be more conserved. Since all the regulatory and coding sequences of the virus were present in the plasmids obtained, complete infectious viral particles were rescued from these clones, and these rescued viral populations were amplified by co-infecting primary embryo liver cells with the rescued virus and the CELO strain of the avian adenovirus type 1. As a proof of concept of the validity of this system for the purpose of gene delivery, recombinant viruses encoding for the LacZ gene as a reporter system were also generated. These recombinant viruses were used to express beta galactosidase activity in primary chicken embryo cell cultures.
Estevez
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Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, 953 College Station Road, Athens, GA 30602, USA.
Villegas
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D000818:Animals; D001483:Base Sequence; D001717:Birds / Q000821:virology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004279:DNA, Viral / Q000737:chemistry* / Q000302:isolation & purification; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D017930:Genes, Reporter; D005814:Genes, Viral; D016679:Genome, Viral; D006801:Humans; D007763:Lac Operon; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011323:Primates / Q000821:virology; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; D001616:beta-Galactosidase