An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing

Aimone, Catherine D.; Hoyer, J. Steen; Dye, Anna; Deppong, David O.; Duffy, Siobain; Carbone, Ignazio; Hanley‐Bowdoin, Linda

doi:10.1016/j.jviromet.2021.114405

Cited by 16 publications

(22 citation statements)

References 42 publications

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“…Viral segment acquisition and transmission rates were estimated and their titers in the source plants, white ies, sucrose, and leaf discs were measured using qPCR. 17 The 48-h IAP allowed the white ies to purge their gut contents of viral components acquired during the AAP.…”

Section: Resultsmentioning

confidence: 99%

“…Inoculations were conducted using low pressure biolistic bombardment to deliver the plasmid DNAs. 17 Only symptomatic plants with infections con rmed by qPCR of viral DNA were used as sources for virus acquisition by white ies.…”

Section: Methodsmentioning

confidence: 99%

“…Copy number (titer) of DNA segments. Quantitative PCR (qPCR) was used to measure the copy numbera of the DNA-A and DNA-B genome segments of ACMV and EACMCV using the primer pairs and conditions described by Aimone et al 2022 17 qPCR plate also contained a standard curve (serial dilution of 3x10 7 to 30 copies of a plasmid DNA containing the viral DNA segment), water control, and negative controls consisting of leaf disk and sucrose sachet samples that had been fed on by white ies that were not exposed to an infected source plant. Ct values were calculated using the Applied Biosystems 7500 Real Time PCR Machine® software.…”

Section: Methodsmentioning

confidence: 99%

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Genome Formula Changes During Whitefly Transmission of Two Bipartite Cassava Mosaic Begomoviruses

Sharpee

Jacobson

et al. 2023

Preprint

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Cassava mosaic disease is caused by a complex of whitefly-transmitted begomovirus species, which often occur in mixed infections. These viruses have bipartite genomes consisting of DNA-A and DNA-B that are encapsidated into separate virions. Individual virus species exist in plants and whitefly vectors as populations comprising both genome segments, which can occur at different frequencies. Both segments are required for infection, and both must be transmitted for virus spread to occur. Cassava plants infected with both cognate segments of African cassava mosaic virus (ACMV) and/or East African cassava mosaic Cameroon virus (EACMCV) were used to examine how titers of the segments in a plant relate to their respective probabilities of acquisition by whiteflies and to the titers of each segment acquired and subsequently transmitted by whiteflies. The relationship between the DNA-A:DNA-B ratio in the plant and the relative likelihood of acquiring each segment differed between ACMV and EACMCV. However, for both viruses, DNA-A:DNA-B ratios acquired by whiteflies differed from those in the source plant and the ratios transmitted by the whitefly were “1” – the ratio at which the highest probability of transmitting both segments is expected.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Genome Formula Changes During Whitefly Transmission of Two Bipartite Cassava Mosaic Begomoviruses

Sharpee

Jacobson

et al. 2023

Preprint

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“…In the past two decades, the NGS technology has been frequently used to assist in the discovery of new virus species [ 71 – 74 ]. In this study, the NGS analysis of the C. morifolium leaves demonstrated the presence of a virus related to the genus Carlavirus within the family Betaflexiviridae [ 11 , 13 ].…”

Section: Discussionmentioning

confidence: 99%

Integrated next-generation sequencing and comparative transcriptomic analysis of leaves provides novel insights into the ethylene pathway of Chrysanthemum morifolium in response to a Chinese isolate of chrysanthemum virus B

Zhong

Yang

et al. 2022

Virol J

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Background Chrysanthemum virus B (CVB), a key member of the genus Carlavirus, family Betaflexiviridae, causes severe viral diseases in chrysanthemum (Chrysanthemum morifolium) plants worldwide. However, information on the mechanisms underlying the response of chrysanthemum plants to CVB is scant. Methods Here, an integrated next-generation sequencing and comparative transcriptomic analysis of chrysanthemum leaves was conducted to explore the molecular response mechanisms of plants to a Chinese isolate of CVB (CVB-CN) at the molecular level. Results In total, 4934 significant differentially expressed genes (SDEGs) were identified to respond to CVB-CN, of which 4097 were upregulated and 837 were downregulated. Gene ontology and functional classification showed that the majority of upregulated SDEGs were categorized into gene cohorts involved in plant hormone signal transduction, phenylpropanoid and flavonoid biosynthesis, and ribosome metabolism. Enrichment analysis demonstrated that ethylene pathway-related genes were significantly upregulated following CVB-CN infection, indicating a strong promotion of ethylene biosynthesis and signaling. Furthermore, disruption of the ethylene pathway in Nicotiana benthamiana, a model plant, using virus-induced gene silencing technology rendered them more susceptible to cysteine-rich protein of CVB-CN induced hypersensitive response, suggesting a crucial role of this pathway in response to CVB-CN infection. Conclusion This study provides evidence that ethylene pathway has an essential role of plant in response to CVB and offers valuable insights into the defense mechanisms of chrysanthemum against Carlavirus.

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“…We saw no obvious patterns of variant co-occurrence across plant samples indicating sample-to-sample cross-contamination with actual virus DNA (Supplementary Tables S2 to S5) but including additional controls (spike-in sequence identifiers or mock-inoculated plant DNA libraries) could rule out or mitigate this possibility. Higher sensitivity for rare variants could potentially be achieved with alternative DNA preparation strategies (Aimone et al 2021b; Pinto et al 2021).…”

Section: Discussionmentioning

confidence: 99%

Rapid multilocus adaptation of clonal cabbage leaf curl virus populations toArabidopsis thaliana

Hoyer

Wilkins

Munshi

et al. 2021

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Cabbage leaf curl virus (CabLCV) has a bipartite single-stranded DNA genome and infects the model plant Arabidopsis thaliana. CabLCV serves as a model for the genus Begomovirus, members of which cause tremendous crop losses worldwide. We have used CabLCV as a model for within-plant virus evolution by inoculating individual plants with infectious clones of both wild-type and mutagenized versions of the CabLCV genome. Consistent with previous reports, detrimental substitutions in the Replication-associated gene (Rep) were readily compensated for by direct reversion and/or alternative mutations. A surprising number of common mutations were detected elsewhere in both viral segments (DNA-A and DNA-B) indicating convergent evolution and suggesting that CabLCV may not be as well adapted to A. thaliana as commonly presumed. Consistent with this idea, a spontaneous coat protein variant consistently rose to higher allele frequency in a hypersusceptible A. thaliana accession (Sei-0) than in another susceptible accession (Col-0). Numerous high-frequency mutations were also detected in a candidate Rep binding site in DNA-B. Our results reinforce the fact that spontaneous mutation of this type of virus occurs rapidly and can change the majority consensus sequence of a within-plant virus population in weeks.

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An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing

Cited by 16 publications

References 42 publications

Genome Formula Changes During Whitefly Transmission of Two Bipartite Cassava Mosaic Begomoviruses

Genome Formula Changes During Whitefly Transmission of Two Bipartite Cassava Mosaic Begomoviruses

Integrated next-generation sequencing and comparative transcriptomic analysis of leaves provides novel insights into the ethylene pathway of Chrysanthemum morifolium in response to a Chinese isolate of chrysanthemum virus B

Rapid multilocus adaptation of clonal cabbage leaf curl virus populations toArabidopsis thaliana

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