RESUMO
Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.
Assuntos
Anticorpos Antivirais , Vírus de Plantas/genética , Vírus de Plantas/imunologia , Vírion/genética , Vírion/imunologia , Animais , Especificidade de Anticorpos , Carlavirus/genética , Carlavirus/imunologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Luteoviridae/genética , Luteoviridae/imunologia , Filogenia , Vírus de Plantas/isolamento & purificação , Potyvirus/genética , Potyvirus/imunologia , RNA Viral/genética , Análise de Sequência de RNA , Solanum tuberosum/virologia , Vírion/isolamento & purificaçãoRESUMO
Large quantities of potato leafroll virus (PLRV) antigen are difficult to obtain because this virus accumulates in plants at a low titer. To overcome this problem, we constructed a binary vector containing chimeric cDNA, in which the coat protein (CP) gene of the crucifer infecting tobacco mosaic virus (crTMV) was substituted for the coat protein gene of PLRV. The PLRV movement protein (MP) gene, which overlaps completely with the CP gene, was doubly mutated to eliminate priming of the PLRV MP translation from ATG codons with no changes to the amino acid sequence of the CP. The untranslated long intergenic region located upstream of the CP gene was removed from the construct. Transcribed powerful tobamovirus polymerase of the produced vector synthesized PLRV CP gene that was, in turn, translated into the protein. CP PLRV packed RNAs from the helical crTMV in spherical virions. Morphology, size and antigenic specificities of the wild-type and chimeric virus were similar. The yield of isolated chimera was about three orders higher than the yield of native PLRV. The genetic manipulations facilitated the generation of antibodies against the chimeric virus, which recognize the wild-type PLRV.
Assuntos
Antígenos Virais/imunologia , Luteoviridae/imunologia , Nicotiana/imunologia , Plantas Geneticamente Modificadas/imunologia , Solanum tuberosum/imunologia , Vírus do Mosaico do Tabaco/imunologia , Proteínas Virais/imunologia , Antígenos Virais/genética , Genoma Viral , Luteoviridae/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/virologia , Biossíntese de Proteínas , RNA Viral , Solanum tuberosum/genética , Solanum tuberosum/virologia , Nicotiana/genética , Nicotiana/virologia , Vírus do Mosaico do Tabaco/genética , Proteínas Virais/genética , Vírion/genética , Vírion/imunologiaRESUMO
Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection.
Assuntos
Interações Hospedeiro-Patógeno , Luteoviridae/imunologia , Luteoviridae/fisiologia , Nicotiana/imunologia , Nicotiana/virologia , Interferência de RNA , Proteínas Virais/metabolismo , Agrobacterium/genética , TransgenesRESUMO
To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the MIA procedure allowed efficient removal of excess sample compounds and reagents. This resulted in lower background values and a higher specificity than a non-wash MIA procedure using conventional beads. In a simplex MIA detection, levels for PVY and PLRV in potato leaf extracts were 10 times lower than ELISA but for PVX 10 timers higher, whereas the specificity was similar. Results of a multiplex assay performed on viruses added to potato leaf extracts were largely similar to those of ELISA for individual viruses. Results of samples infected naturally with PVX, PVY or PLRV were comparable with ELISA.
