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Agarose gel analysis of rolling circle‐amplified (RCA) DNA. Undigested (lanes 1, 4, and 5), KpnI‐digested (lane 2), BamHI‐digested (lane 3), and PstI‐digested (lane 6) RCA products derived from BSUIV (lanes 1 and 2) and BSUMV (lanes 3, 4, 5, and 6) infected banana plants. M, 1 kb ladder from Invitrogen
Source publication
Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae.
Badnaviral sequences found in banana are distributed over three main clades of the
genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome
of many plants, including banana, is invaded by numerous badnaviral sequences
although badnaviru...
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Le virus de la mosaïque en tirets des bananiers ou Banana streak virus (BSV) décrit en Côte d'Ivoire en 1958 affecte la croissance du bananier, le rendement et la qualité des fruits et constitue une entrave à l'échange de germoplasmes. L'objectif de cette étude est d'évaluer la prévalence de deux isolats de BSV (BSOLV et BSGFV) sur les bananiers pl...
Citations
... Besides protein coding regions, badnaviruses are expected to contain a tRNA-Met binding sequence that defines the beginning of the viral genome, which was identified as 5ʹ-TGGTAGCGAAGCAAGGTTCA-3ʹ; a TATA-box upstream of the tRNA Met binding, identified at positions 6,806-6,812 as 5ʹ-TATATAA-3ʹ; and a polyadenylation signal, which was located in the intergenic region at positions 6,520-6,525 with sequence 5ʹ-AATAAA-3ʹ (Figure 4). Given the evidence, we believe this virus to be a novel badnavirus species for which we propose the name B. passiflorae following the latest virus nomenclature guidelines approved by the International Committee on Taxonomy of Viruses (Walker et al., 2021) Pararetroviruses can be integrated into the host genome through a mechanism probably involving non-homologous recombination that can lead to nonfunctional translation products (Chabannes et al., 2020). A more detailed comparison of B. passiflorae with contig Pe93N7 suggests an ancient integration event into the P. edulis genome, as three segments with significant similarity to portions of the CP (S1), protease (S2), and RNase H domains were identified at positions 31, 469-32,397, 32,427-33,327, and 33,373-33,667, arranged in the order S2, S3, S1, and with an inversion in the S2 segment (Figure 4). ...
Purple passion fruit, one of the most important fruit exports in Colombia, has been seriously affected in recent years by several viral diseases. The aim of the present study was to investigate the virome of asymptomatic purple passion fruit samples from commercial plots and nurseries in Antioquia (Colombia). The presence of viruses was first tested by high-throughput sequencing (HTS) of bulked leaf samples using total RNA and dsRNA inputs. Interestingly, the HTS data suggested the presence of a new badnavirus with a genome of 6,951 bp, which was confirmed by DNA sequencing of rolling circle amplification products. This virus has been tentatively named Badnavirus passiflorae as it is the first report of a badnavirus infecting passion fruit. The presence of Soybean mosaic virus (SMV), Cowpea aphid-borne mosaic virus (CABMV), Cucumber mosaic virus (CMV), and Passion fruit yellow mosaic virus (PFYMV) was further investigated using RT-qPCR, as well as evaluated the infection by begomoviruses and badnaviruses through PCR. Using these techniques, B. passiflorae was detected in all the samples, PFYMV in 20% of adult plants and 10% of plantlets, and SMV in 20% of adult plants and 30% of plantlets. None of the samples tested positive for CABMV, CMV, or begomoviruses.
Background
High-throughput sequencing (HTS) technologies completed by the bioinformatic analysis of the generated data are becoming an important detection technique for virus diagnostics. They have the potential to replace or complement the current PCR-based methods thanks to their improved inclusivity and analytical sensitivity, as well as their overall good repeatability and reproducibility. Cross-contamination is a well-known phenomenon in molecular diagnostics and corresponds to the exchange of genetic material between samples. Cross-contamination management was a key drawback during the development of PCR-based detection and is now adequately monitored in routine diagnostics. HTS technologies are facing similar difficulties due to their very high analytical sensitivity. As a single viral read could be detected in millions of sequencing reads, it is mandatory to fix a detection threshold that will be informed by estimated cross-contamination. Cross-contamination monitoring should therefore be a priority when detecting viruses by HTS technologies.
Results
We present Cont-ID, a bioinformatic tool designed to check for cross-contamination by analysing the relative abundance of virus sequencing reads identified in sequence metagenomic datasets and their duplication between samples. It can be applied when the samples in a sequencing batch have been processed in parallel in the laboratory and with at least one specific external control called Alien control. Using 273 real datasets, including 68 virus species from different hosts (fruit tree, plant, human) and several library preparation protocols (Ribodepleted total RNA, small RNA and double-stranded RNA), we demonstrated that Cont-ID classifies with high accuracy (91%) viral species detection into (true) infection or (cross) contamination. This classification raises confidence in the detection and facilitates the downstream interpretation and confirmation of the results by prioritising the virus detections that should be confirmed.
Conclusions
Cross-contamination between samples when detecting viruses using HTS (Illumina technology) can be monitored and highlighted by Cont-ID (provided an alien control is present). Cont-ID is based on a flexible methodology relying on the output of bioinformatics analyses of the sequencing reads and considering the contamination pattern specific to each batch of samples. The Cont-ID method is adaptable so that each laboratory can optimise it before its validation and routine use.
High-throughput sequencing (HTS) technologies have brought tremendous improvements in the ability to detect plant viruses and have great potential for application in virus routine diagnostics. The performance criteria of an HTS test need therefore to be estimated and compared with traditional virus indexing tests before it can be used in routine diagnostics. In this study, 78 Musa accessions previously indexed for viruses by molecular tests and/or electron microscopy were tested individually or in pools using an HTS protocol based on total RNA sequencing. The analytical sensitivity of HTS and RT-PCR was also compared by independent testing on serial dilutions of RNA extracts. In total, 136 libraries were sequenced in five batches, and the sequences were analyzed for virus detection. The external alien control, a wheat sample infected by barley yellow dwarf virus, monitored the contamination burden and determined an adaptative detection threshold. Overall, the HTS test displayed a better analytical sensitivity than the RT-PCR and a better inclusivity than the classical indexing protocol, as distant isolates and new viral species were only detected by the HTS test. The repeatability and reproducibility of virus detection were both 100%, although differences in number of sequencing reads per virus were observed between replicates. The diagnostic sensitivity was very high, but false positive results were observed. Finally, the results also underlined the need for expert judgement in the interpretation of the results. In conclusion, the HTS test with an alien control and completed by expert evaluation fulfilled the criteria of the virus indexing protocol for Musa germplasm.
[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
The genome sequence of a novel circular DNA virus related to members of the genus Badnavirus was identified in diseased jujube trees by high-throughput sequencing and verified by conventional Sanger sequencing of cloned PCR products. The name "jujube badnavirus WS" (JuBWS) is proposed for this virus. Diseased jujube leaves showed yellow mosaic and malformation symptoms, with round chlorotic spots found on diseased fruit. The genome of this virus has a length of 6450 nt and has a typical badnavirus genomic structure with three open reading frames (ORFs). JuBWS was identified as a novel badnavirus based on nucleotide differences in the RNase (RT + RNase H) coding region of ORF3. The JuBWS sequence showed 70.48–76.41% nucleotide sequence identity to other known badnaviruses, thus meeting the taxonomic criterion for establishing a new species within the genus Badnavirus. This study suggested that the novel badnavirus might be a pathogen associated with jujube mosaic disease, and this will be investigated in the future.
Cocoa, Theobroma cacao, is an important tropical perennial crop grown widely in the humid tropics. The exchange of cocoa germplasm between germplasm collections and breeding centres is vital for varietal development. Intermediate quarantine facilities, such as the International Cocoa Quarantine Centre, Reading UK (ICQC-R) play a vital role in ensuring the transfer of germplasm whilst minimising the risk of spreading pests and diseases. Current screening procedures combine visual inspection and molecular techniques, which are effective in detecting Cocoa swollen shoot virus (CSSV), a badnavirus, which causes severe losses but are restricted to West Africa. However, the detection of latent or mild virus infections that produce no visual symptoms has been a challenge. Recently two badnavirus species of cocoa producing mild symptoms, cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV), have been sequenced. Here, we report new assays for the detection of these two species, for the first time in non-symptomatic accessions. Evolutionary and bioinformatic analyses of the viruses suggest their most recent source was from Trinidad, though there is historic evidence that these viruses may have their origin in South America and then become widespread globally over the last century. We also report a novel colorimetric Loop-mediated isothermal amplification (LAMP) assay for the detection of CYVBV. This simple and accurate method could be employed in field virus testing.
Theobroma cacao is one of the most economically important tropical trees, being the source of chocolate. As part of an ongoing study to understand the diversity of the badnavirus complex, responsible for the cacao swollen shoot virus disease in West Africa, evidence was found recently of virus-like sequences in asymptomatic cacao plants. The present study exploited the wealth of genomic resources in this crop, and combined bioinformatic, molecular, and genetic approaches to report for the first time the presence of integrated badnaviral sequences in most of the cacao genetic groups. These sequences, which we propose to name eTcBV for endogenous T. cacao bacilliform virus, varied in type with each predominating in a specific genetic group. A diagnostic multiplex PCR method was developed to identify the homozygous or hemizygous condition of one specific insert, which was inherited as a single Mendelian trait. These data suggest that these integration events occurred before or during the species diversification in Central and South America, and prior to its cultivation in other regions. Such evidence of integrated sequences is relevant to the management of cacao quarantine facilities and may also aid novel methods to reduce the impact of such viruses in this crop.
