scholarly journals Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta)

2012 ◽  
Vol 93 (12) ◽  
pp. 2668-2681 ◽  
Author(s):  
Karyna Rosario ◽  
Anisha Dayaram ◽  
Milen Marinov ◽  
Jessica Ware ◽  
Simona Kraberger ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Molecular Techniques ◽  
Limited Information ◽  
Dna Viruses ◽  
Rolling Circle ◽  
Viral Genomes ◽  
Initiator Protein ◽  
Circular Ssdna ◽  
Insect Populations ◽  
Ssdna Viruses

Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

A variety of highly divergent eukaryotic ssDNA viruses in sera of pigs

2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Caroline Tochetto ◽  
Samuel Paulo Cibulski ◽  
Ana Paula Muterle Varela ◽  
Cristine Cerva ◽  
Diane Alves de Lima ◽  
...  
Keyword(s):  
Potential Role ◽  
Clinical Signs ◽  
Viral Metagenomics ◽  
Dna Viruses ◽  
Circular Dna ◽  
Viral Genomes ◽  
The Family ◽  
Circular Ssdna ◽  
Ssdna Viruses ◽  
Targeted Approach

Over the last decade, viral metagenomics has been established as a non-targeted approach for identifying viruses in stock animals, including pigs. This has led to the identification of a vast diversity of small circular ssDNA viruses. The present study focuses on the investigation of eukaryotic circular Rep-encoding single-stranded (CRESS) DNA viral genomes present in serum of commercially reared pigs from southern Brazil. Several CRESS DNA viral genomes were detected, including representatives of the families Smacoviridae (n=5), Genomoviridae (n=3), Redondoviridae (n=1), Nenyaviridae (n=1) and other yet unclassified genomes (n=9), plus a circular DNA molecule, which probably belongs to the phylum Cressdnaviricota. A novel genus within the family Smacoviridae, tentatively named ‘Suismacovirus’, comprising 21 potential new species, is proposed. Although the reported genomes were recovered from pigs with clinical signs of respiratory disease, further studies should examine their potential role as pathogens. Nonetheless, these findings highlight the diversity of circular ssDNA viruses in serum of domestic pigs, expand the knowledge on CRESS DNA viruses’ genetic diversity and distribution and contribute to the global picture of the virome of commercially reared pigs.

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

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 Infectious Molecular Clones of Adeno-Associated Virus Isolated Directly from Human Tissues

2008 ◽  
Vol 83 (3) ◽  
pp. 1456-1464 ◽  
Author(s):  
Bruce C. Schnepp ◽  
Ryan L. Jensen ◽  
K. Reed Clark ◽  
Philip R. Johnson
Keyword(s):  
Unit Length ◽  
Rolling Circle Amplification ◽  
Inverted Terminal Repeat ◽  
Human Tissues ◽  
Adeno Associated Virus ◽  
Host Chromosome ◽  
Rolling Circle ◽  
Viral Genomes ◽  
Natural Hosts

ABSTRACT Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.

scholarly journals Composite Analysis of the Virome and Bacteriome of HIV/HPV Co-Infected Women Reveals Proxies for Immunodeficiency

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 422 ◽  
Author(s):  
Juliana Siqueira ◽  
Gislaine Curty ◽  
Deng Xutao ◽  
Cristina Hofer ◽  
Elizabeth Machado ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Hpv Infection ◽  
Cervical Lesions ◽  
Dna Viruses ◽  
Circular Dna ◽  
Rolling Circle ◽  
Cell Counts ◽  
Increased Risk ◽  
High Risk Hpv ◽  
Ngs Data

The human cervical microbiome is complex, and its role in health and disease has just begun to be elucidated. In this study, 57 cervical swab samples from 19 HIV/HPV co-infected women were analyzed for both virome and bacteriome composition. Virome analysis focused on circular DNA viruses through rolling circle amplification followed by next-generation sequencing (NGS). Data were assigned to virus families and genera, and HPV types were identified. NGS data of bacterial 16S from a subset of 24 samples were assigned to operational taxonomic units and classified according to vaginal microbiome community state types (CSTs). Four viral families were found: Papillomaviridae, Anelloviridae, Genomoviridae, and Herpesviridae. Papillomavirus reads were more abundant in women with premalignant cervical lesions, which were also strongly associated with multiple (≥3) high-risk HPV infection. Anellovirus read abundance was negatively correlated with host CD4+ T-cell counts. The bacteriome revealed the presence of CST III and CST IV, and women with ≥1% frequency of genomovirus or herpesvirus reads displayed an increased risk of carrying CST IV. By characterizing the composition of the cervical circular DNA viruses and the bacteriome of HIV/HPV co-infected women, we identified putative interactions between these two microorganism communities and their associations with patients’ clinical characteristics, notably immunodeficiency status.

scholarly journals The genetic diversity of “papillomavirome” in bovine teat papilloma lesions

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Jéssica Tatiane Sauthier ◽  
Cíntia Daudt ◽  
Flavio Roberto Chaves da Silva ◽  
Christian Diniz Beduschi Travassos Alves ◽  
Fabiana Quoos Mayer ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
High Throughput Sequencing ◽  
Molecular Detection ◽  
Bos Taurus ◽  
Rolling Circle Amplification ◽  
Dna Viruses ◽  
Rolling Circle ◽  
Double Stranded Dna ◽  
Wide Range ◽  
Study Support

Abstract Background Papillomaviruses are small nonenveloped, circular double-stranded DNA viruses that belong to the Papillomaviridae family. To date, 29 Bos taurus papillomavirus (BPV) types have been described. Studies involving mixed BPV infections have rarely been reported in contrast to human papillomavirus (HPV), which is commonly described in numerous studies showing coinfections. Moreover, previous studies had shown that HPV coinfections increase the risk of carcinogenesis. In the present study, we used rolling-circle amplification followed by a high-throughput sequencing (RCA-HTS) approach in 23 teat papillomas from southern Brazil. Results Eleven well-characterized BPV types and 14 putative new BPV types were genetically characterized into the Xi, Epsilon and Dyoxipapillomavirus genera according to phylogenetic analysis of the L1 gene, which expands the previous 29 BPV types to 43. Moreover, BPV coinfections were detected in the majority (56.3%) of the papilloma lesions analyzed, suggesting a genetic diverse “papillomavirome” in bovine teat warts. Conclusions The data generated in this study support the possibility that a wide range of BPV is probably underdetected by conventional molecular detection tools, and that BPV coinfections are underestimated and probably genetic diverse. Additionally, 14 new BPV types were characterized, increasing the knowledge regarding BPV genetic diversity.

scholarly journals Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2585 ◽  
Author(s):  
Victoria M. Pearson ◽  
S. Brian Caudle ◽  
Darin R. Rokyta
Keyword(s):  
Wastewater Treatment ◽  
Microbial Communities ◽  
Rolling Circle Amplification ◽  
Treatment Plant ◽  
Microbial Pathogens ◽  
Model Organisms ◽  
Single Stranded Dna ◽  
Dna Viruses ◽  
Depth Analysis ◽  
Ssdna Viruses

Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (CircoviridaeandGeminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such asCircoviridae,Geminiviridae, andMicroviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group.

scholarly journals Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2777 ◽  
Author(s):  
Simon Roux ◽  
Natalie E. Solonenko ◽  
Vinh T. Dang ◽  
Bonnie T. Poulos ◽  
Sarah M. Schwenck ◽  
...  
Keyword(s):  
Multiple Displacement Amplification ◽  
Ecosystem Functions ◽  
Library Preparation ◽  
Single Stranded Dna ◽  
Preparation Methods ◽  
Dna Viruses ◽  
Viral Genomes ◽  
Sequencing Library ◽  
Dsdna Viruses ◽  
Ssdna Viruses

BackgroundViruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation).MethodsHere we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses.ResultsMock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from theMicroviridaefamily, can be among the most abundant viral genomes in a sample.DiscussionTogether these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems.

scholarly journals High global diversity of cycloviruses amongst dragonflies

2013 ◽  
Vol 94 (8) ◽  
pp. 1827-1840 ◽  
Author(s):  
Anisha Dayaram ◽  
Kristen A. Potter ◽  
Angela B. Moline ◽  
Dana Drake Rosenstein ◽  
Milen Marinov ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Sequence Data ◽  
Genome Structure ◽  
Sister Group ◽  
Regulatory Elements ◽  
Intergenic Regions ◽  
The Usa ◽  
Circular Ssdna ◽  
Insect Populations ◽  
Ssdna Viruses

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genus Cyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra- and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution.

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