title: A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses.
abstract: Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules.
authors.affiliation: 1] Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Área de Genética, Campus de Teatinos, 29071 Málaga, Spain [2].
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Rodríguez-Negrete
authors.affiliation: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain.
authors.forename: Sonia
authors.initials: S
authors.lastname: Sánchez-Campos
authors.affiliation: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain.
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Cañizares
authors.affiliation: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Navas-Castillo
authors.affiliation: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Estación Experimental "La Mayora", 29750 Algarrobo-Costa, Málaga, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Moriones
authors.affiliation: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Área de Genética, Campus de Teatinos, 29071 Málaga, Spain.
authors.forename: Eduardo R
authors.initials: ER
authors.lastname: Bejarano
authors.affiliation: Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Científicas, (IHSM-UMA-CSIC) Área de Genética, Campus de Teatinos, 29071 Málaga, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Grande-Pérez
mesh_terms: D000818:Animals; D053839:Begomovirus / Q000235:genetics / Q000472:pathogenicity; D018076:DNA, Complementary / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D017791:Geminiviridae / Q000235:genetics / Q000472:pathogenicity; D006801:Humans; D018551:Solanum lycopersicum / Q000235:genetics / Q000821:virology; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D018515:Plant Leaves / Q000821:virology; D014771:Virion / Q000235:genetics* / Q000472:pathogenicity; D014779:Virus Replication / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D004277:DNA, Single-Stranded