scholarly journals Identification of a Novel Geminivirus in Fraxinus rhynchophylla in Korea

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2385
Author(s):  
Aamir Lal ◽  
Yong-Ho Kim ◽  
Thuy Thi Bich Vo ◽  
I Gusti Ngurah Prabu Wira Sanjaya ◽  
Phuong Thi Ho ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Indian Subcontinent ◽  
Chemical Constituents ◽  
Rolling Circle Amplification ◽  
Open Reading Frames ◽  
Stem Loop ◽  
Rolling Circle ◽  
Reading Frame ◽  
Infectious Clones ◽  
Ash Trees

Fraxinus rhynchophylla, common name ash, belongs to the family Oleaceae and is found in China, Korea, North America, the Indian subcontinent, and eastern Russia. It has been used as a traditional herbal medicine in Korea and various parts of the world due to its chemical constituents. During a field survey in March 2019, mild vein thickening (almost negligible) was observed in a few ash trees. High-throughput sequencing of libraries of total DNA from ash trees, rolling-circle amplification (RCA), and polymerase chain reaction (PCR) allowed the identification of a Fraxinus symptomless virus. This virus has five confirmed open reading frames along with a possible sixth open reading frame that encodes the movement protein and is almost 2.7 kb in size, with a nonanucleotide and stem loop structure identical to begomoviruses. In terms of its size and structure, this virus strongly resembles begomoviruses, but does not show any significant sequence identity with them. To confirm movement of the virus within the trees, different parts of infected trees were examined, and viral movement was successfully observed. No satellite molecules or DNA B were identified. Two-step PCR confirmed the virion and complementary strands during replication in both freshly collected infected samples of ash tree and Nicotiana benthamiana samples agro-inoculated with infectious clones. This taxon is so distantly grouped from other known geminiviruses that it likely represents a new geminivirus genus.

scholarly journals Characterization of Two Novel Polyomaviruses of Birds by Using Multiply Primed Rolling-Circle Amplification of Their Genomes

2006 ◽  
Vol 80 (7) ◽  
pp. 3523-3531 ◽  
Author(s):  
Reimar Johne ◽  
Walter Wittig ◽  
Daniel Fernández-de-Luco ◽  
Ursula Höfle ◽  
Hermann Müller
Keyword(s):  
Rolling Circle Amplification ◽  
Large Tumor ◽  
Rolling Circle ◽  
Reading Frame ◽  
Field Samples ◽  
Close Relationship ◽  
Avian Polyomavirus ◽  
Avian Group ◽  
Pyrrhula Pyrrhula

ABSTRACT Polyomaviruses are small nonenveloped particles with a circular double-stranded genome, approximately 5 kbp in size. The mammalian polyomaviruses mainly cause persistent subclinical infections in their natural nonimmunocompromised hosts. In contrast, the polyomaviruses of birds—avian polyomavirus (APV) and goose hemorrhagic polyomavirus (GHPV)—are the primary agents of acute and chronic disease with high mortality rates in young birds. Screening of field samples of diseased birds by consensus PCR revealed the presence of two novel polyomaviruses in the liver of an Eurasian bullfinch (Pyrrhula pyrrhula griseiventris) and in the spleen of a Eurasian jackdaw (Corvus monedula), tentatively designated as finch polyomavirus (FPyV) and crow polyomavirus (CPyV), respectively. The genomes of the viruses were amplified by using multiply primed rolling-circle amplification and cloned. Analysis of the FPyV and CPyV genome sequences revealed a close relationship to APV and GHPV, indicating the existence of a distinct avian group among the polyomaviruses. The main characteristics of this group are (i) involvement in fatal disease, (ii) the existence of an additional open reading frame in the 5′ region of the late mRNAs, and (iii) a different manner of DNA binding of the large tumor antigen compared to that of the mammalian polyomaviruses.

scholarly journals Molecular Analysis of pDL10 from Acidianus ambivalens Reveals a Family of Related Plasmids from Extremely Thermophilic and Acidophilic Archaea

Genetics ◽  
1999 ◽  
Vol 152 (4) ◽  
pp. 1307-1314
Author(s):  
Arnulf Kletzin ◽  
Angelika Lieke ◽  
Tim Urich ◽  
Robert L Charlebois ◽  
Christoph W Sensen
Keyword(s):  
Amino Acid ◽  
Regulatory Protein ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Mung Bean Nuclease ◽  
Stem Loop ◽  
Rolling Circle ◽  
Rep Protein ◽  
Homologous Protein ◽  
Rep Proteins

Abstract The 7598-bp plasmid pDL10 from the extremely thermophilic, acidophilic, and chemolithoautotrophic Archaeon Acidianus ambivalens was sequenced. It contains 10 open reading frames (ORFs) organized in five putative operons. The deduced amino acid sequence of the largest ORF (909 aa) showed similarity to bacterial Rep proteins known from phages and plasmids with rolling-circle (RC) replication. From the comparison of the amino acid sequences, a novel family of RC Rep proteins was defined. The pDL10 Rep protein shared 45-80% identical residues with homologous protein genes encoded by the Sulfolobus islandicus plasmids pRN1 and pRN2. Two DNA regions capable of forming extended stem-loop structures were also conserved in the three plasmids (48-69% sequence identity). In addition, a putative plasmid regulatory protein gene (plrA) was found, which was conserved among the three plasmids and the conjugative Sulfolobus plasmid pNOB8. A homolog of this gene was also found in the chromosome of S. solfataricus. Single-stranded DNA of both pDL10 strands was detected with a mung bean nuclease protection assay using PCR detection of protected fragments, giving additional evidence for an RC mechanism of replication.

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.

scholarly journals A Novel Virus Detected in Papillomas and Carcinomas of the Endangered Western Barred Bandicoot (Perameles bougainville) Exhibits Genomic Features of both the Papillomaviridae and Polyomaviridae

2007 ◽  
Vol 81 (24) ◽  
pp. 13280-13290 ◽  
Author(s):  
Lucy Woolford ◽  
Annabel Rector ◽  
Marc Van Ranst ◽  
Andrea Ducki ◽  
Mark D. Bennett ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Open Reading Frames ◽  
Nonstructural Proteins ◽  
T Antigens ◽  
Virus Family ◽  
Rolling Circle ◽  
Large Genome Size ◽  
Novel Virus ◽  
L1 And L2

ABSTRACT Conservation efforts to prevent the extinction of the endangered western barred bandicoot (Perameles bougainville) are currently hindered by a progressively debilitating cutaneous and mucocutaneous papillomatosis and carcinomatosis syndrome observed in captive and wild populations. In this study, we detected a novel virus, designated the bandicoot papillomatosis carcinomatosis virus type 1 (BPCV1), in lesional tissue from affected western barred bandicoots using multiply primed rolling-circle amplification and PCR with the cutaneotropic papillomavirus primer pairs FAP59/FAP64 and AR-L1F8/AR-L1R9. Sequencing of the BPCV1 genome revealed a novel prototype virus exhibiting genomic properties of both the Papillomaviridae and the Polyomaviridae. Papillomaviral properties included a large genome size (∼7.3 kb) and the presence of open reading frames (ORFs) encoding canonical L1 and L2 structural proteins. The genomic organization in which structural and nonstructural proteins were encoded on different strands of the double-stranded genome and the presence of ORFs encoding the nonstructural proteins large T and small t antigens were, on the other hand, typical polyomaviral features. BPCV1 may represent the first member of a novel virus family, descended from a common ancestor of the papillomaviruses and polyomaviruses recognized today. Alternatively, it may represent the product of ancient recombination between members of these two virus families. The discovery of this virus could have implications for the current taxonomic classification of Papillomaviridae and Polyomaviridae and can provide further insight into the evolution of these ancient virus families.

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.

scholarly journals Transcription Strategy in a Closterovirus: a Novel 5′-Proximal Controller Element of Citrus Tristeza Virus Produces 5′- and 3′-Terminal Subgenomic RNAs and Differs from 3′ Open Reading Frame Controller Elements

2003 ◽  
Vol 77 (1) ◽  
pp. 340-352 ◽  
Author(s):  
Siddarame Gowda ◽  
María A. Ayllón ◽  
Tatineni Satyanarayana ◽  
Moshe Bar-Joseph ◽  
William O. Dawson
Keyword(s):  
Promoter Activity ◽  
Citrus Tristeza Virus ◽  
Open Reading Frames ◽  
Stem Loop ◽  
Reading Frame ◽  
Subgenomic Rnas ◽  
Modular Evolution ◽  
Different Origins ◽  
Tristeza Virus ◽  
Citrus Tristeza

ABSTRACT Citrus tristeza virus (CTV) produces more than thirty 3′- or 5′-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5′-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3′ genes deleted produce only the larger LMT1 RNAs. These 5′-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5′TR) surrounding the putative termination sites (nt ∼550 to 1000) was duplicated in the 3′ end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5′-terminal plus-strand sgRNA, here much larger (∼11 kb), apparently by termination. Surprisingly, a new 3′-terminal sgRNA was observed from the duplicated 5′TR. A large 3′-terminal sgRNA resulting from the putative promoter activity of the native 5′TR was not observed, possibly because of the down-regulation of a promoter ∼19 kb from the 3′ terminus. However, we were able to observe a sgRNA produced from the native 5′TR of a small defective RNA, which placed the native 5′TR closer to the 3′ terminus, demonstrating sgRNA promoter activity of the native 5′TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5′TR (in the 3′ position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3′- and 5′-terminal sgRNAs. These mutations, when introduced in frame in the native 5′TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3′-terminal sgRNA while having no impact on replication, demonstrating that neither 5′- nor 3′-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5′TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3′ open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3′-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.

scholarly journals Identification of Chili leaf curl virus Causing Leaf Curl Disease of Petunia in Oman

Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 572-572 ◽  
Author(s):  
A. A. Al-Shihi ◽  
S. Akhtar ◽  
A. J. Khan
Keyword(s):  
Indian Subcontinent ◽  
Rolling Circle Amplification ◽  
Rapid Evolution ◽  
Post Inoculation ◽  
Multiple Sequence ◽  
Rolling Circle ◽  
Begomovirus Genome ◽  
Leaf Curl Virus ◽  
Leaf Curl Disease ◽  
Leaf Curl

Petunias (Petunia × hybrida) are the most important ornamental plants in Oman. In 2012, petunias were observed in public parks and airport landscape in Dhofar region with symptoms of upward leaf curling, yellowing and vein clearing, and size reduction in leaves. Almost all plants in the surveyed landscape showed high infestation of Bemisia tabaci and symptoms that suggested infection with a begomovirus. Six symptomatic samples were collected from three different sites. All symptomatic samples were found PCR-positive with diagnostic primers for begomovirus (3) when DNA extracted from infected leaves was used as template. Nucleic acids extracted from the symptomatic leaves were used to amplify circular DNA molecules by rolling circle amplification method. The amplified concatameric products were digested with restriction enzyme PstI, which yielded a product ∼2.8 kb in size. The putative begomovirus fragment was cloned and sequenced in both orientations. Partial sequences of six clones were 99 to 100% similar and thus only two clones, PT-2 and PT-3, were fully sequenced. The whole genomes of both clones were 2,761 bp, and both were deposited in GenBank under accession numbers HF968755 and HF968756 for the isolates PT-2 and PT-3, respectively. Both sequences had six open reading frames; Rep, TrAP, REn, and C4 genes in complementary sense; and CP and V2 genes in virion-sense, typical of the begomovirus genome organization. Upon alignment, the two sequences showed 99.4% nucleotide identity with each other, thus representing isolates of a single begomovirus species. BlastN comparison showed PT-2 and PT-3 from petunia were 94 to 95% identical to the sequences of ChCLV from Oman (JN604490 to JN604500), which were obtained from other hosts. ClustalV multiple sequence alignment showed that isolates PT-2 and PT-3 shared maximum sequence identity of 93.3 and 92.8%, respectively, with an isolate of ChLCV-OM (JN604495). According to ICTV rules for begomoviruses, PT-3 should be considered to be a new strain of ChLCV-OM and PT-2 a variant of the already existing ChLCV-OM strain. We propose the name for this new strain as the “Petunia strain” of Chili leaf curl virus (ChLCV-Pet). Two infectious clones were constructed from the PT-2 and PT-3 sequences, clones as 1.75-genome sequences in a binary vector, suitable for agroinfection to confirm their infectivity. Both clones, PT-2 and PT-3, produced typical leaf curl disease symptoms upon inoculation on petunia 18 days post inoculation. The presence of the same virus in symptomatic field infected and inoculated petunia was confirmed by Southern blot using 650 bp DIG labeled probe prepared from CP region of PT-3 isolate. ChLCV-OM, a monopartite begomovirus, is widely associated with leaf curl disease of tomato and pepper in Oman, with its origin traced to the Indian subcontinent (2). Identification of a new strain of ChLCV from petunia provides evidence of an ongoing rapid evolution of begomoviruses in this region. Although petunia has been tested as an experimental host for some begomoviruses (1,4), this is the first report of petunia as natural host for ChLCV, a begomovirus previously reported in tomato and pepper in Oman. References: (1) Cui et al. J. Virol. 78:13966, 2004. (2) Khan et al. Virus Res. 177:87, 2013. (3) Khan et al. Plant Dis. 97:1396, 2013. (4) Urbino et al. Arch. Virol. 149:417, 2003.

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 Blueberry fruit drop-associated virus: A New Member of the Family Caulimoviridae Isolated From Blueberry Exhibiting Fruit-Drop Symptoms

Plant Disease ◽  
2016 ◽  
Vol 100 (11) ◽  
pp. 2211-2214 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
Robert R. Martin
Keyword(s):  
Rolling Circle Amplification ◽  
The United States ◽  
Washington State ◽  
Cloning And Sequencing ◽  
Rolling Circle ◽  
Reading Frame ◽  
Fruit Drop ◽  
Perfect Correlation ◽  
The Family ◽  
Single Field

This study describes the nucleotide sequence and genome organization of a new DNA virus isolated from ‘Bluecrop’ blueberry plants exhibiting fruit-drop symptoms and named Blueberry fruit drop-associated virus (BFDaV). Blueberry fruit drop disease was first detected in blueberry plants in British Columbia, Canada in the late 1990s, and in a single field in northern Washington state in the United States in 2012. Infected bushes abort nearly 100% of their fruit about three weeks prior to harvest, when the berries are about 3 to 5 mm in diameter. At harvest, the affected plants appear taller than healthy ones as there is no fruit weighing down the branches. The virus was amplified from diseased material using rolling circle amplification, followed by enzyme digestion, cloning, and sequencing. The full genome of BFDaV is 9,850 bp in length and contains a single open reading frame, encoding for a polyprotein, and a large noncoding region. Based on the genome size and organization and phylogenetics, BFDaV is proposed as a new and the largest member of family Caulimoviridae. Finally, in mapping part of a field with fruit-drop symptoms, there was a nearly perfect correlation between the presence of the virus and fruit-drop symptoms.

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