scholarly journals Discovery of a novel Torque teno sus virus species: genetic characterization, epidemiological assessment and disease association

2012 ◽  
Vol 93 (12) ◽  
pp. 2682-2691 ◽  
Author(s):  
Vivian Cornelissen-Keijsers ◽  
Alexandra Jiménez-Melsió ◽  
Denny Sonnemans ◽  
Martí Cortey ◽  
Joaquim Segalés ◽  
...  
Keyword(s):  
Genetic Characterization ◽  
Phylogenetic Analyses ◽  
Rolling Circle Amplification ◽  
Disease Association ◽  
Virus Species ◽  
Viral Dna ◽  
Rolling Circle ◽  
Viral Loads ◽  
Wasting Syndrome ◽  
Torque Teno Sus Virus

The study describes a novel Torque teno sus virus (TTSuV) species, provisionally named Torque teno sus virus k2b (TTSuVk2b), originally found in commercial pig sera by applying the rolling-circle amplification technique. Full-length sequences of TTSuVk2b were obtained, annotated and used in the phylogenetic analyses, which revealed that TTSuVk2b is a novel Anellovirus species within the genus Kappatorquevirus of the family Anelloviridae. Quantitative PCR techniques were developed to determine total TTSuV DNA quantities as well as the prevalence and viral DNA quantities of TTSuV1, TTSuVk2a and TTSuVk2b. The mean total TTSuV load in seven commercial sera was determined at 6.3 log10 DNA copies ml−1 of serum, with TTSuVk2b loads being the lowest at 4.5 log10 DNA copies ml−1 of serum. Subsequently, prevalence and loads of TTSuVs were determined in pig sera from 17 countries. TTSuVk2b prevalence ranged from 0 to 100 % with viral loads from 3.3 to 4.6 log10 copies ml−1 of sera. TTSuVk2a, so far the only species in the genus Kappatorquevirus, has been linked to an economically important swine disease, namely post-weaning multisystemic wasting syndrome (PMWS). Considering the grouping of TTSuVk2b in the same genus as TTSuVk2a, TTSuVk2b prevalence and viral DNA load were determined in PMWS-affected animals and healthy counterparts. This revealed that TTSuVk2a and TTSuVk2b are not only genetically related, but also that their viral loads in serum are elevated in PMWS animals compared with those of healthy pen mates. In summary, the present work describes a novel TTSuV species including its genetic characterization, epidemiological assessment and potential disease association.

scholarly journals An improved experimental pipeline for preparing circular ssDNA viruses for next-generation sequencing

2020 ◽  
Author(s):  
Catherine D. Aimone ◽  
J. Steen Hoyer ◽  
Anna E. Dye ◽  
David O. Deppong ◽  
Siobain Duffy ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rolling Circle Amplification ◽  
Next Generation ◽  
Viral Dna ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Short Read ◽  
Rolling Circle ◽  
Optimized Protocol ◽  
Generation Sequencing

AbstractWe present an optimized protocol for enhanced amplification and enrichment of viral DNA for Next Generation Sequencing of begomovirus genomes. The rapid ability of these viruses to evolve threatens many crops and underscores the importance of using next generation sequencing efficiently to detect and understand the diversity of these viruses. We combined enhanced rolling circle amplification (RCA) with EquiPhi29 polymerase and size selection to generate a cost-effective, short-read sequencing method. This optimized protocol produced short-read sequencing with at least 50% of the reads mapping to the viral reference genome. We provide other insights into common misconceptions about RCA and lessons we have learned from sequencing single-stranded DNA viruses. Our protocol can be used to examine viral DNA as it moves through the entire pathosystem from host to vector, providing valuable information for viral DNA population studies, and would likely work well with other CRESS DNA viruses.HighlightsProtocol for short-read, high throughput sequencing of single-stranded DNA viruses using random primersComparison of the sequencing of total DNA versus size-selected DNAComparison of phi29 and Equiphi29 DNA polymerases for rolling circle amplification of viral single-stranded DNA genomes

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.

Rolling-circle amplification of viral DNA genomes using phi29 polymerase

2009 ◽  
Vol 17 (5) ◽  
pp. 205-211 ◽  
Author(s):  
Reimar Johne ◽  
Hermann Müller ◽  
Annabel Rector ◽  
Marc van Ranst ◽  
Hans Stevens
Keyword(s):  
Rolling Circle Amplification ◽  
Viral Dna ◽  
Rolling Circle

scholarly journals Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Tomasz Krzywkowski ◽  
Sibel Ciftci ◽  
Farzaneh Assadian ◽  
Mats Nilsson ◽  
Tanel Punga
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Single Cell ◽  
Cell Population ◽  
Rolling Circle Amplification ◽  
Expression Patterns ◽  
Human Adenovirus ◽  
Viral Dna ◽  
Rolling Circle ◽  
Infected Cells

ABSTRACT An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells. IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

Sensitive and selective viral DNA detection assay via microbead-based rolling circle amplification

2008 ◽  
Vol 18 (22) ◽  
pp. 5871-5874 ◽  
Author(s):  
Eric Schopf ◽  
Nicholas O. Fischer ◽  
Yong Chen ◽  
Jeffrey B.-H. Tok
Keyword(s):  
Dna Detection ◽  
Rolling Circle Amplification ◽  
Viral Dna ◽  
Rolling Circle ◽  
Detection Assay

scholarly journals Cloning, Structural Characterization, and Phylogenetic Analysis of Flower MADS-Box Genes from Crocus (Crocus sativusL.)

2007 ◽  
Vol 7 ◽  
pp. 1047-1062 ◽  
Author(s):  
Athanasios S. Tsaftaris ◽  
Alexios N. Polidoros ◽  
Konstantinos Pasentsis ◽  
Apostolos Kalivas
Keyword(s):  
Molecular Mechanisms ◽  
Phylogenetic Analyses ◽  
Rolling Circle Amplification ◽  
Crocus Sativus ◽  
Flower Formation ◽  
Mads Box ◽  
Crop Species ◽  
Rolling Circle ◽  
Cdna Sequences ◽  
Single Compound

Crocus (Crocus sativusL.) is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals) and three tepals in whorl 2 (inner tepals). The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation.Here we review the different methods followed or developed for obtaining these sequences involving conventional 5' 3' RACE, as well as newly developed methods from our group, named Rolling Circle Amplification – RACE (RCA-RACE) and its modification named familyRCA-RACE (famRCA-RACE). Furthermore, the characteristics of the protein structure and their common and specific domains for each type of MADS-box transcription factors in this lower nongrass monocot belonging to the Iridaceae family are described. Finally, a phylogenetic tree of all the MADS-box sequences available in our lab is presented and discussed in relation to other data from studies of species of the Iridaceae group and closely related families from an evolutionary perspective. The structural and phylogenetic analyses are based on both published and unpublished data.

Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) viral loads in serum of postweaning multisystemic wasting syndrome (PMWS)-affected and healthy pigs in Brazil

2015 ◽  
Vol 101 ◽  
pp. 38-41 ◽  
Author(s):  
Thais Fumaco Teixeira ◽  
Samuel Paulo Cibulski ◽  
Helton Fernandes dos Santos ◽  
Adriéli Wendlant ◽  
Francisco Esmaile de Sales Lima ◽  
...  
Keyword(s):  
Postweaning Multisystemic Wasting Syndrome ◽  
Viral Loads ◽  
Wasting Syndrome ◽  
Torque Teno Sus Virus

scholarly journals Genomic characterization of ten novel cutaneous human papillomaviruses from keratotic lesions of immunosuppressed patients

2011 ◽  
Vol 92 (7) ◽  
pp. 1585-1594 ◽  
Author(s):  
Anja Köhler ◽  
Marc Gottschling ◽  
Kizzie Manning ◽  
Mandy D. Lehmann ◽  
Eric Schulz ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Rolling Circle Amplification ◽  
Organ Transplant ◽  
Human Papillomaviruses ◽  
Rolling Circle ◽  
Non Melanoma Skin Cancer ◽  
Immunosuppressed Patients ◽  
Genomic Characterization ◽  
Hpv Types

Viral warts from immunosuppressed organ transplant recipients (OTR) persist over years and may progress into non-melanoma skin cancer. The types of human papillomaviruses (HPV) in such lesions are different from that seen in the general population. A subset of these lesions is not infected with the classical wart-associated HPV types. In order to gain a better understanding of the HPV types in those lesions, we isolated ten novel HPVs from persisting keratotic lesions of immunosuppressed OTRs by rolling circle amplification and subsequent long-template PCR. Additionally, we sequenced and characterized the whole genome of the ten novel HPV types. Phylogenetic analyses revealed that nine HPV types belonged to the genus Gammapapillomavirus (γ-PV) and one to the genus Betapapillomavirus. In a phylogenetic analysis using L1 fragments of human and non-human PV types, primate papillomaviruses and our novel HPV types nested within the genus γ-PV in a highly polyphyletic pattern. This study significantly broadens the knowledge concerning the diversity and evolution of the poorly known γ-PV types.

Ferromagnetic Resonance Biosensor for Homogeneous and Volumetric Detection of DNA

2017 ◽  
Author(s):  
Bo Tian ◽  
Peter Svedlindh ◽  
Mattias Strömberg ◽  
Erik Wetterskog
Keyword(s):  
Magnetic Nanoparticles ◽  
Ferromagnetic Resonance ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rolling Circle Amplification ◽  
Resonance Field ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Serum Samples ◽  
Rolling Circle

In this work, we demonstrate for the first time, a ferromagnetic resonance (FMR) based homogeneous and volumetric biosensor for magnetic label detection. Two different isothermal amplification methods, <i>i.e.</i>, rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) are adopted and combined with a standard electron paramagnetic resonance (EPR) spectrometer for FMR biosensing. For RCA-based FMR biosensor, binding of RCA products of a synthetic Vibrio cholerae target DNA sequence gives rise to the formation of aggregates of magnetic nanoparticles. Immobilization of nanoparticles within the aggregates leads to a decrease of the net anisotropy of the system and a concomitant increase of the resonance field. A limit of detection of 1 pM is obtained with an average coefficient of variation of 0.16%, which is superior to the performance of other reported RCA-based magnetic biosensors. For LAMP-based sensing, a synthetic Zika virus target oligonucleotide is amplified and detected in 20% serum samples. Immobilization of magnetic nanoparticles is induced by their co-precipitation with Mg<sub>2</sub>P<sub>2</sub>O<sub>7</sub> (a by-product of LAMP) and provides a detection sensitivity of 100 aM. The fast measurement, high sensitivity and miniaturization potential of the proposed FMR biosensing technology makes it a promising candidate for designing future point-of-care devices.<br>

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