RESUMEN
Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.
Asunto(s)
Infecciones por Adenoviridae/veterinaria , Adenoviridae/aislamiento & purificación , Heces/virología , Tracto Gastrointestinal/virología , Hominidae/virología , Macaca/virología , Adenoviridae/genética , Infecciones por Adenoviridae/virología , África , Animales , Animales de Zoológico , Genes Virales , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Recombinación GenéticaRESUMEN
The successful use of any adenoviral vectors is predicated upon the use of a serotype that is not neutralized by circulating antibodies. However, efforts to develop a diverse repertoire of serologically distinct adenovirus vectors may be hindered by the necessity to generate cell lines to allow for the successful propagation of vectors deleted of essential genes. A strategy to construct chimeric adenoviruses whereby the rescue and propagation of an E1-deleted HAdV-B-derived adenoviral vector can be achieved using existing cell lines such as HEK 293 is reported. It is further shown that this strategy may be more widely applicable.
Asunto(s)
Adenovirus de los Simios/genética , ADN Viral/genética , Vectores Genéticos , Genoma Viral , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/inmunología , Proteínas E4 de Adenovirus/genética , Proteínas E4 de Adenovirus/inmunología , Adenovirus de los Simios/clasificación , Adenovirus de los Simios/inmunología , Animales , Línea Celular , Estudios de Factibilidad , Humanos , Modelos Genéticos , Pruebas de Neutralización , Pan troglodytes/virología , Plásmidos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Serotipificación , TransfecciónRESUMEN
Functional complementation of cellular defects has been a valuable approach for localizing causative genes to specific chromosomes. The complementation strategy was followed by positional cloning and characterization of genes for their biological relevance. We herein describe strategies used for the construction of monochromosomal hybrids and their applications for cloning and characterization of genes related to cell growth, cell senescence and DNA repair. We have cloned RNaseT2, GluR6 (glutamate ionotropic receptor kainate type subunit 2-GRIK2) and protein tyrosine phosphatase, receptor type K (PTPRK) genes using these strategies.
Asunto(s)
Cromosomas Humanos/genética , Técnicas de Transferencia de Gen , Células Híbridas/metabolismo , Modelos Genéticos , Animales , Fusión Celular , Línea Celular , Humanos , Células Híbridas/citología , Receptores de Ácido Kaínico/genética , Ribonucleasas/genética , Proteínas Supresoras de Tumor/genética , Receptor de Ácido Kaínico GluK2RESUMEN
The ability to regulate both the timing and specificity of gene expression mediated by viral vectors will be important in maximizing its utility. We describe the development of an adeno-associated virus (AAV)-based vector with tissue-specific gene regulation, using the ARGENT dimerizer-inducible system. This two-vector system based on AAV serotype 9 consists of one vector encoding a combination of reporter genes from which expression is directed by a ubiquitous, inducible promoter and a second vector encoding transcription factor domains under the control of either a heart- or liver-specific promoter, which are activated with a small molecule. Administration of the vectors via either systemic or intrapericardial injection demonstrated that the vector system is capable of mediating gene expression that is tissue specific, regulatable, and reproducible over induction cycles. Somatic gene transfer in vivo is being considered in therapeutic applications, although its most substantial value will be in basic applications such as target validation and development of animal models.