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Descripción
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| The number of viral genomes infecting a cell (or multiplicity of cellular infection, MOI) determines the extent of such phenomena as recombination between viral strains, competition and complementation of defective mutants. These interactions are major players in the evolution and epidemiology of viruses. Despite its importance, estimations of MOI during plant virus infections are limited to three examples, a DNA virus (Califlower mosaic virus, CaMV) and two RNA viruses (Tobacco mosaic virus, TMV and Soil-borne wheat mosaic virus, SBWMV). MOI values in these three studies varied widely, ranging from 1 to 13 over the course of the viral infection in the host plant. Small values of MOI indicate mechanisms preventing co-infection of cells. Tomato bushy stunt virus (TBSV) is the type member of the genus Tombusvirus. The genome of TBSV is a positive sense ssRNA of approximately 4.8 kb which encodes five open reading frames: two proteins involved in replication, the coat protein, the movement protein and a suppressor of gene silencing. Under high multiplicity serial passages, the infection of TBSV (and other Tombusvirus) is associated with the appearing of defective interfering RNAs (DI-RNAs). DI-RNAs are incomplete RNA viral genomes which require the present of their helper virus for replication and packaging. The presence of DI-RNAs results in a decreasing of virus titer and usually in an attenuation of the symptoms caused by the helper virus. The high titer of DI-RNAs suggests that mechanisms excluding co-infection with the helper virus could not apply. In this work we have estimated MOI during the infection of TBSV in the systemic host Nicotiana benthamiana, with or without the presence of DI-RNA molecules in the initial inoculum. Our purpose was to evaluate the possible variation in MOI levels due to the interaction of the helper virus and derived DI-RNAs, coexisting in the same plant cell. For this purpose, we have adopted two technical approaches. On the one hand, we have used two fluorescent tagged viruses to quantify the number of single and double infected cells, which allows the estimation of genotype frequencies and MOI during the viral infection. On the other hand, we have developed a strategy based on single-cell quantitative PCR following by high-resolution melting (HRM) to quantify single and mixed infected cells with TBSV and the associated DI-RNA, DI-72, both labeled with two different genetic markers introduced by molecular engineering. | |
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Internacional
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No |
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Nombre congreso
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XII Congreso Nacional de Virología |
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Tipo de participación
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960 |
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Lugar del congreso
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Burgos |
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Revisores
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Si |
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ISBN o ISSN
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00-0000-00 |
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DOI
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Fecha inicio congreso
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09/06/2013 |
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Fecha fin congreso
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12/06/2013 |
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Desde la página
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334 |
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Hasta la página
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334 |
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Título de las actas
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Virología |