scholarly journals Viromes of Ten Alfalfa Plants in Australia Reveal Diverse Known Viruses and a Novel RNA Virus

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 214
Author(s):  
Samira Samarfard ◽  
Alistair R. McTaggart ◽  
Murray Sharman ◽  
Nicolás E. Bejerman ◽  
Ralf G. Dietzgen
Keyword(s):  
Complete Genome ◽  
High Throughput Sequencing ◽  
Rna Viruses ◽  
Sequence Data ◽  
Rna Virus ◽  
Rolling Circle Amplification ◽  
French Bean ◽  
Cdna Libraries ◽  
Restriction Enzyme Digestion ◽  
Rolling Circle

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa.

scholarly journals A workflow for accurate metabarcoding using nanopore MinION sequencing

2020 ◽  
Author(s):  
Bilgenur Baloğlu ◽  
Zhewei Chen ◽  
Vasco Elbrecht ◽  
Thomas Braukmann ◽  
Shanna MacDonald ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Data ◽  
Rolling Circle Amplification ◽  
Error Rates ◽  
Read Length ◽  
Taxonomic Assignment ◽  
Major Drawback ◽  
Rolling Circle ◽  
Sequencing Platform ◽  
Sequencing Platforms

AbstractMetabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short-read high-throughput sequencing platforms. The Oxford Nanopore MinION™, a portable sequencing platform, represents a low-cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%.To test if the MinION™ represents a viable alternative to other sequencing platforms we used rolling circle amplification (RCA) to generate full-length consensus DNA barcodes (658bp of cytochrome oxidase I - COI) for a bulk mock sample of 50 aquatic invertebrate species. By applying two different laboratory protocols, we generated two MinION™ runs that were used to build consensus sequences. We also developed a novel Python pipeline, ASHURE, for processing, consensus building, clustering, and taxonomic assignment of the resulting reads.We were able to show that it is possible to reduce error rates to a median accuracy of up to 99.3% for long RCA fragments (>45 barcodes). Our pipeline successfully identified all 50 species in the mock community and exhibited comparable sensitivity and accuracy to MiSeq. The use of RCA was integral for increasing consensus accuracy, but it was also the most time-consuming step during the laboratory workflow and most RCA reads were skewed towards a shorter read length range with a median RCA fragment length of up to 1262bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding but we recommend the exploration of other isothermal amplification procedures to improve consensus length.

scholarly journals Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yuka Torii ◽  
Kazuhiro Horiba ◽  
Satoshi Hayano ◽  
Taichi Kato ◽  
Takako Suzuki ◽  
...  
Keyword(s):  
Kawasaki Disease ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Data ◽  
Early Stage ◽  
Rna Virus ◽  
Systemic Vasculitis ◽  
Human Herpesvirus ◽  
Acute Stage ◽  
Serum Samples

Abstract Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

scholarly journals Plant Virus Genome Is Shaped by Specific Dinucleotide Restrictions That Influence Viral Infection

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alfonso González de Prádena ◽  
Adrián Sánchez Jimenez ◽  
David San León ◽  
Peter Simmonds ◽  
Juan Antonio García ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
High Throughput Sequencing ◽  
Rna Viruses ◽  
Rna Virus ◽  
Virus Genome ◽  
Viral Fitness ◽  
Plum Pox Virus ◽  
Dependent Manner ◽  
Viral Restriction ◽  
Rna Accumulation

ABSTRACT The presence of CpG and UpA dinucleotides is restricted in the genomes of animal RNA viruses to avoid specific host defenses. We wondered whether a similar phenomenon exists in nonanimal RNA viruses. Here, we show that these two dinucleotides, especially UpA, are underrepresented in the family Potyviridae, the most important group of plant RNA viruses. Using plum pox virus (PPV; Potyviridae family) as a model, we show that an increase in UpA frequency strongly diminishes virus accumulation. Remarkably, unlike previous observations in animal viruses, PPV variants harboring CpG-rich fragments display just faint (or no) attenuation. The anticorrelation between UpA frequency and viral fitness additionally demonstrates the relevance of this particular dinucleotide: UpA-high mutants are attenuated in a dose-dependent manner, whereas a UpA-low variant displays better fitness than its parental control. Using high-throughput sequencing, we also show that UpA-rich PPV variants are genetically stable, without apparent changes in sequence that revert and/or compensate for the dinucleotide modification despite its attenuation. In addition, we also demonstrate here that the PPV restriction of UpA-rich variants works independently of the classical RNA silencing pathway. Finally, we show that the anticorrelation between UpA frequency and RNA accumulation applies to mRNA-like fragments produced by the host RNA polymerase II. Together, our results inform us about a dinucleotide-based system in plant cells that controls diverse RNAs, including RNA viruses. IMPORTANCE Dinucleotides (combinations of two consecutive nucleotides) are not randomly present in RNA viruses; in fact, the presence of CpG and UpA is significantly repressed in their genomes. Although the meaning of this phenomenon remains obscure, recent studies with animal-infecting viruses have revealed that their low CpG/UpA frequency prevents virus restriction via a host antiviral system that recognizes, and promotes the degradation of, CpG/UpA-rich RNAs. Whether similar systems act in organisms from other life kingdoms has been unknown. To fill this gap in our knowledge, we built several synthetic variants of a plant RNA virus with deoptimized dinucleotide frequencies and analyzed their viral fitness and genome adaptation. In brief, our results inform us for the first time about an effective dinucleotide-based system that acts in plants against viruses. Remarkably, this viral restriction in plants is reminiscent of, but not identical to, the equivalent antiviral response in animals.

scholarly journals Identification and Characterization of Two Novel Geminiviruses Associated with Paper Mulberry (Broussonetia papyrifera) Leaf Curl Disease

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 3010-3018 ◽  
Author(s):  
Yuanjian Qiu ◽  
Song Zhang ◽  
Haodong Yu ◽  
Zhiyou Xuan ◽  
Liu Yang ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Close Association ◽  
Phylogenetic Analyses ◽  
Rolling Circle Amplification ◽  
Field Investigation ◽  
Broussonetia Papyrifera ◽  
New Members ◽  
Rolling Circle ◽  
Mulberry Leaf ◽  
Leaf Curl

Paper mulberry (Broussonetia papyrifera) is a perennial woody plant used as source material for Cai Lun paper making, in traditional Chinese medicine, and as livestock feed. To identify the presence of viruses in paper mulberry plants affected by a disease with leaf curl symptoms, high-throughput sequencing of total RNA was performed. Analysis of transcriptome libraries allowed the reconstruction of two geminivirus-like genomes. Rolling-circle amplification and PCR with back-to-back primers confirmed the presence of two geminiviruses with monopartite genomes in these plants, with the names paper mulberry leaf curl virus 1 and 2 (PMLCV-1 and PMLCV-2) proposed. The genomes of PMLCV-1 (3,056 nt) and PMLCV-2 (3,757 to 3,763 nt) encode six proteins, with the V4 protein of PMLCV-1 and the V3 proteins of both viruses having low similarities to any known protein in databases. Alternative splicing of an intron, akin to that of mastre-, becurto-, capula-, and grabloviruses, was identified by small RNA (sRNA)-seq and RNA-seq reads mapping to PMLCV-1 and PMLCV-2 antisense transcripts. Phylogenetic analyses and pairwise comparisons showed that PMLCV-1 and PMLCV-2 are most closely related to, but distinct from, two unassigned geminiviruses, citrus chlorotic dwarf associated virus and mulberry mosaic dwarf associated virus, suggesting that they are two new members of the family Geminiviridae. Field investigation confirmed the close association of the two viruses with leaf curl symptoms in paper mulberry plants and that coinfection can aggravate the symptoms.

Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome

2020 ◽  
Vol 160 (6) ◽  
pp. 329-334
Author(s):  
Alfredo Diaz-Lara ◽  
Nola J. Mosier ◽  
Kristian Stevens ◽  
Karen E. Keller ◽  
Robert R. Martin
Keyword(s):  
High Throughput Sequencing ◽  
Rolling Circle Amplification ◽  
Standard Test ◽  
Molecular Techniques ◽  
Test Method ◽  
Plant Genome ◽  
Red Raspberry ◽  
Rolling Circle ◽  
Certification Programs ◽  
Dnase Treatment

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis “Munger” has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on “Munger”, suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia

2021 ◽  
Author(s):  
Yafei Tang ◽  
Zhenggang Li ◽  
Xiaoman She ◽  
Lin Yu ◽  
Guobing Lan ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Pcr Amplification ◽  
Monopartite Begomoviruses ◽  
Yellow Vein ◽  
Structure Characteristic ◽  
Restriction Enzyme Digestion ◽  
Monopartite Begomovirus ◽  
Rolling Circle ◽  
Ageratum Yellow Vein Virus ◽  
Malvastrum Coromandelianum

Abstract A previously undescribed monopartite begomovirus was identified from Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomoviruses, in Kampot province, Cambodia. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling circle amplification and restriction enzyme digestion. The genome of the virus was 2,737 nucleotides in length (KP188831), and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nt identities of 87.7% with Ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification with the betasatellite-specific primer pair β01/β02. The satellite molecule (1,346 nt, KP188832) had a structure characteristic like other betasatellites associated with begomoviruses, and shared the highest nt identity of 84.8% with Malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses ( Geminiviridae ) and betasatellites ( Tolecusatellitidae ), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus species，for which the name Malvastrum yellow vein Cambodia virus (MaYVCV) is proposed, whereas, the betasatellite is identified as an previously undescribed novel betasatellite species, for which the name Malvastrum yellow vein Cambodia batesatellite (MaYVKHB) is proposed.

scholarly journals A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification

2004 ◽  
Vol 78 (10) ◽  
pp. 4993-4998 ◽  
Author(s):  
Annabel Rector ◽  
Ruth Tachezy ◽  
Marc Van Ranst
Keyword(s):  
Restriction Enzyme ◽  
Rolling Circle Amplification ◽  
Enzyme Analysis ◽  
Amplification Efficiency ◽  
Sequence Information ◽  
Restriction Enzyme Digestion ◽  
Hpv 16 ◽  
Circular Dna ◽  
Rolling Circle ◽  
Papillomavirus Dna

ABSTRACT The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 104-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.

scholarly journals Application of a sequence-based taxonomic classification method to uncultured and unclassified marine single-stranded RNA viruses in the order Picornavirales

2019 ◽  
Vol 5 (2) ◽  
Author(s):  
Marli Vlok ◽  
Andrew S Lang ◽  
Curtis A Suttle
Keyword(s):  
Rna Viruses ◽  
Sequence Data ◽  
Rna Virus ◽  
Taxonomic Classification ◽  
Sound Classification ◽  
Virus Diversity ◽  
Species Demarcation Threshold ◽  
The Family ◽  
Genetic Features ◽  
Taxonomic Framework

Abstract Metagenomics has altered our understanding of microbial diversity and ecology. This includes its applications to viruses in marine environments that have demonstrated their enormous diversity. Within these are RNA viruses, many of which share genetic features with members of the order Picornavirales; yet, very few of these have been taxonomically classified. The only recognized family of marine RNA viruses is the Marnaviridae, which was founded based on discovery and characterization of the species Heterosigma akashiwo RNA virus. Two additional genera of marine RNA viruses, Labyrnavirus (one species) and Bacillarnavirus (three species), were subsequently defined within the order Picornavirales but not assigned to a family. We have defined a sequence-based framework for taxonomic classification of twenty marine RNA viruses into the family Marnaviridae. Using RNA-dependent RNA polymerase (RdRp) phylogeny and distance-based analyses, we assigned the genera Labyrnavirus and Bacillarnavirus to the family Marnaviridae and created four additional genera in the family: Locarnavirus (four species), Kusarnavirus (one species), Salisharnavirus (four species) and Sogarnavirus (six species). We used pairwise capsid protein comparisons to delineate species within families, with 75 per cent identity as the species demarcation threshold. The family displays high sequence diversities and Jukes–Cantor distances for both the RdRp and capsid genes, suggesting that the classified viruses are not representative of all of the virus diversity within the family and that there are many more extant taxa. Our proposed taxonomic framework provides a sound classification system for this group of viruses that will have broadly applicable principles for other viral groups. It is based on sequence data alone and provides a robust taxonomic framework to include viruses discovered via metagenomic studies, thereby greatly expanding the realm of viruses subject to taxonomic classification.

scholarly journals First Report of Ageratum enation virus, Betasatellite and Alphasatellite Causing Leaf Curl and Enation Disease of Amaranthus hypochondriacus in India

Plant Disease ◽  
2014 ◽  
Vol 98 (9) ◽  
pp. 1285-1285 ◽  
Author(s):  
A. Srivastava ◽  
S. Kumar ◽  
S. K. Raj
Keyword(s):  
Sequence Data ◽  
Disease Incidence ◽  
Rolling Circle Amplification ◽  
Nucleotide Identity ◽  
Post Inoculation ◽  
Specific Primers ◽  
Rolling Circle ◽  
Amaranthus Hypochondriacus ◽  
Close Phylogenetic Relationship ◽  
Leaf Curl

During a survey in February 2011, severe symptoms of upward leaf curling, vein enation on lower side of the leaves, and shortening of internodes were observed on 20 out of 117 Amaranthus hypochondriacus plants (17% disease incidence) examined at breeding plots of CSIR-NBRI, Lucknow. These symptoms are typical of begomovirus infection. PCR with begomovirus-specific primers (3) produced the expected ~1.1-kb product from DNA extracts of 20 symptomatic plants but not from a non-symptomatic plant, suggesting the association of a begomovirus. The full-length begomoviral genome from a representative sample was amplified by rolling circle amplification using Ø-29 DNA polymerase and digested by BamHI, which resulted in a ~2.7 kb product when electrophoresed in 1.0% agarose gel. The product obtained was cloned, sequenced, and sequence data of 2,753 nucleotides was deposited in GenBank (Accession No. JF682242). BLASTn analysis revealed 97 to 98% nucleotide identity and forms a distinct clade with Ageratum enation virus (AEV) isolates. This shows the virus in A. hypochondriacus to be an isolate of AEV. The separate PCRs were also performed with betasatellite and alphasatellite specific primers (1,2) that resulted in ~1.3-kb amplicons from all samples, suggesting their association. The amplification products were cloned and sequenced. An analysis of betasatellite (JX512904) revealed highest 98% nucleotide identity and close phylogenetic relationship with Ageratum leaf curl betasatellite (ALCB, JQ710745). The alphasatellite (JX512905) showed highest 95% identity and close relationship with Hibiscus leaf curl alphasatellite (HLCA, FN794199). This shows the betasatellite and alphasatellite in A. hypochondriacus to be isolates of ALCB and HLCA, respectively. The partial direct repeat clones of the begomovirus (pCAM-AEV), betasatellite (pCAM-ALCB), alphasatellite (pCAM-HLCA) were generated and mobilized into Agrobacterium tumefaciens strain GV3101 and infiltrated in A. hypochondriacus seedlings. The plants inoculated with pCAM-AEV, pCAM-ALCB, and pCAM-HLCA; pCAM-AEV and pCAM-ALCB developed severe leaf curl and enation symptoms on 5/5 plant at 35 days post inoculation, which were similar to those of naturally infected plants, satisfying Koch's postulates. On the other hand, plants inoculated with pCAM-AEV alone or in combination with pCAM-HLCA developed mild symptoms. Plants inoculated with pCAM-ALCB and pCAM-HLCA did not develop symptoms. The results here show that leaf curl and enation disease of A. hypochondriacus in India is caused by AEV and ALCB and that an alphasatellite may be associated with symptomatic plants. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2) S. E. Bull et al. Mol. Biotechnol. 23:83, 2003. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

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