A real-time RT-PCR assay for quantifying the fitness of tobacco etch virus in competition experiments

Carrasco, Purificación; Daròs, José‐Antonio; Patrícia, Ana; Elena, Santiago F.

doi:10.1016/j.jviromet.2006.09.020

Cited by 81 publications

(99 citation statements)

References 22 publications

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“…Transcripts were precipitated (1.5 vol of DEPC-treated water, 1.5 vol of 7.5 m LiCl, 50 mm EDTA), collected, and resuspended in DEPC-treated water (Carrasco et al 2007). RNA integrity was assessed by gel electrophoresis and its concentration spectrophotometrically determined using a Biophotometer (Eppendorf).…”

Section: Virus and Plantsmentioning

confidence: 99%

“…A. Darò s (unpublished results) have estimated that, on average, an infected cell yields 1555 genomes (quantified by real-time quantitative RT-PCR). To estimate the number of generations experienced by TEV at the time points where the samples were taken, we revisited previously published data on the kinetics of TEV accumulation (Carrasco et al 2007). Reanalyzing these data, we found that the model that better describes TEV accumulation within an infected plant was a four-parameters Gompertz growth equation (R 2 ¼ 0.975) (Campbell and Madden 1990).…”

Section: Virus and Plantsmentioning

confidence: 99%

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The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

Tromas

Elena

2010

Genetics

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Knowing mutation rates and the molecular spectrum of spontaneous mutations is important to understanding how the genetic composition of viral populations evolves. Previous studies have shown that the rate of spontaneous mutations for RNA viruses widely varies between 0.01 and 2 mutations per genome and generation, with plant RNA viruses always occupying the lower side of this range. However, this peculiarity of plant RNA viruses is based on a very limited number of studies. Here we analyze the spontaneous mutational spectrum and the mutation rate of Tobacco etch potyvirus, a model system of positive sense RNA viruses. Our experimental setup minimizes the action of purifying selection on the mutational spectrum, thus giving a picture of what types of mutations are produced by the viral replicase. As expected for a neutral target, we found that transitions and nonsynonymous (including a few stop codons and small deletions) mutations were the most abundant type. This spectrum was notably different from the one previously described for another plant virus. We have estimated that the spontaneous mutation rate for this virus was in the range 10 À6 À10À5 mutations per site and generation. Our estimates are in the same biological ballpark that previous values reported for plant RNA viruses. This finding gives further support to the idea that plant RNA viruses may have lower mutation rates than their animal counterparts.T HE rate of spontaneous mutation is a key parameter to understanding the genetic structure of populations over time. Mutation represents the primary source of genetic variation on which natural selection and genetic drift operate. Although the exact value of the mutation rate is important for several evolutionary theories, accurate estimates are available only for a handful of organisms. RNA viruses show mutation rates that are orders of magnitude higher than those of their DNA-based hosts and in the range of 0

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Section: Virus and Plantsmentioning

confidence: 99%

Section: Virus and Plantsmentioning

confidence: 99%

The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

Tromas

Elena

2010

Genetics

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“…The fitness of various isolates can be estimated by a measure of replication rate and competitiveness (Chao, 1990;Holland et al, 1991). Recent technologies such as quantitative PCR (qPCR) allow the accurate measurement of viral RNA concentration during in vivo studies, based on which the relative copy numbers of viral genomes can be estimated (Weber et al, 2003;Van Maarseveen et al, 2006;Carrasco et al, 2007a). In the present paper, these specific quantification tools (e.g.…”

Section: Introductionmentioning

confidence: 99%

“…All statistical analyses, including analysis of variance (ANOVA), a Student-Newman-Keuls test and a t-test, were performed using the GLM procedure of SAS version 8.01 (SAS Institute Inc.). As described in Carrasco et al (2007a), the fitness, W, of the different genotypes relative to the reference genotypes PVY KRED and PVY N was calculated according to the formula:…”

Section: Introductionmentioning

confidence: 99%

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants

Rolland

Kerlan

Jacquot

2009

Journal of General Virology

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The prevalence of necrotic potato virus Y (PVY) in natural populations could reflect increased fitness of necrotic isolates. In this paper, the effects of the acquisition of molecular determinants (A/G 2213 and A/C 2271 ) involved in necrosis capacity on both the number of progeny produced and the competitiveness of PVY were characterized. The relationship between necrosis and fitness was tested using (i) Nicotiana tabacum cv. competitiveness. In contrast, nucleotides involved in necrotic properties were associated with decreased fitness. Finally, in the host that did not respond to infection with necrosis, the benefit associated with the PVY N -605 genetic background was higher than the cost associated with the acquisition of molecular determinants involved in necrosis capacity. The opposite result was obtained in the host responding to the infection with necrosis. These results indicate that the emergence of necrotic isolates from a non-necrotic population is unlikely in tobacco.

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“…These viruses include Chilli veinal mottle virus (ChiVMV; Hwang et al, 2009), Colombian datura virus (CDV; Salamon and Palkovics, 2005), Tobacco etch virus (TEV; Carrasco et al, 2007), Chrysanthemum stem necrosis virus (CSNV; Bezerra et al, 1999), and Iris yellow spot virus (IYSV; Pappu et al, 2006) (Table 1). ChiVMV, CDV and TEV are positivestrand RNA viruses and consist of one RNA segment belonging to the genus Potyvirus, family Potyviridae (Table 1).…”

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confidence: 99%

RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

Lee¹,

Cho²,

Choi³

et al. 2011

The Plant Pathology Journal

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In order to detect quarantine plant viruses, we developed reverse transcription-polymerase chain reaction (RT-PCR) primer pairs for five single-stranded (ss) plant RNA viruses that are not currently reported in Korea but could be potential harmful plant viral pathogens. Three viruses such as Chilli veinal mottle virus (ChiVMV), Colombian datura virus (CDV), and Tobacco etch virus (TEV) belong to the genus Potyvirus while Chrysanthemum stem necrosis virus (CSNV) and Iris yellow spot virus (IYSV) are members of the genus Tospovirus. To design RT-PCR primers, we used reported gene sequences corresponding to the capsid protein and polyprotein for ChiVMV, CDV, and TEV while using nucleocapsid protein regions for CSNV and IYSV. At least two different primer pairs were designed for each virus. Fifteen out of 16 primer pairs were successfully applied in detection of individual quarantine virus with high specificity and efficiency. Taken together, this study provides a rapid and useful protocol for detection of five quarantine viruses.

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A real-time RT-PCR assay for quantifying the fitness of tobacco etch virus in competition experiments

Cited by 81 publications

References 22 publications

The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

The acquisition of molecular determinants involved in potato virus Y necrosis capacity leads to fitness reduction in tobacco plants

RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

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