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 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 Adintoviruses: A Proposed Animal-Tropic Family of Midsize Eukaryotic Linear dsDNA (MELD) Viruses

2020 ◽  
Author(s):  
Gabriel J Starrett ◽  
Michael J Tisza ◽  
Nicole L Welch ◽  
Anna K Belford ◽  
Alberto Peretti ◽  
...  
Keyword(s):  
Genome Packaging ◽  
Metagenomic Sequence ◽  
Dna Viruses ◽  
Diverse Range ◽  
Integrase Gene ◽  
Data Mining Approach ◽  
Double Stranded Dna ◽  
Wide Range ◽  
Linear Dsdna ◽  
Dsdna Viruses

Abstract Polintons (also known as Mavericks) were initially identified as a widespread class of eukaryotic transposons named for their hallmark type B DNA polymerase and retrovirus-like integrase genes. It has since been recognized that many polintons encode possible capsid proteins and viral genome-packaging ATPases similar to those of a diverse range of double-stranded DNA (dsDNA) viruses. This supports the inference that at least some polintons are actually viruses capable of cell-to-cell spread. At present, there are no polinton-associated capsid protein genes annotated in public sequence databases. To rectify this deficiency, we used a data-mining approach to investigate the distribution and gene content of polinton-like elements and related DNA viruses in animal genomic and metagenomic sequence datasets. The results define a discrete family-like clade of viruses with two genus-level divisions. We propose the family name Adintoviridae, connoting similarities to adenovirus virion proteins and the presence of a retrovirus-like integrase gene. Although adintovirus-class PolB sequences were detected in datasets for fungi and various unicellular eukaryotes, sequences resembling adintovirus virion proteins and accessory genes appear to be restricted to animals. Degraded adintovirus sequences are endogenized into the germlines of a wide range of animals, including humans.

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.

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 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.

scholarly journals Impacts of Genome-Wide Analyses on Our Understanding of Human Herpesvirus Diversity and Evolution

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Daniel W. Renner ◽  
Moriah L. Szpara
Keyword(s):  
High Throughput Sequencing ◽  
Evolutionary Dynamics ◽  
Human Herpesvirus ◽  
Ribosome Profiling ◽  
Genomic Diversity ◽  
Rna Seq ◽  
Dna Viruses ◽  
Double Stranded Dna ◽  
Genome Wide ◽  
Human Herpesviruses

ABSTRACTUntil fairly recently, genome-wide evolutionary dynamics and within-host diversity were more commonly examined in the context of small viruses than in the context of large double-stranded DNA viruses such as herpesviruses. The high mutation rates and more compact genomes of RNA viruses have inspired the investigation of population dynamics for these species, and recent data now suggest that herpesviruses might also be considered candidates for population modeling. High-throughput sequencing (HTS) and bioinformatics have expanded our understanding of herpesviruses through genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures. Here we discuss recent data on the mechanisms that generate herpesvirus genomic diversity and underlie the evolution of these virus families. We focus on human herpesviruses, with key insights drawn from veterinary herpesviruses and other large DNA virus families. We consider the impacts of cell culture on herpesvirus genomes and how to accurately describe the viral populations under study. The need for a strong foundation of high-quality genomes is also discussed, since it underlies all secondary genomic analyses such as RNA sequencing (RNA-Seq), chromatin immunoprecipitation, and ribosome profiling. Areas where we foresee future progress, such as the linking of viral genetic differences to phenotypic or clinical outcomes, are highlighted as well.

scholarly journals Virulent Poxviruses Inhibit DNA Sensing by Preventing STING Activation

2018 ◽  
Vol 92 (10) ◽  
Author(s):  
Iliana Georgana ◽  
Rebecca P. Sumner ◽  
Greg J. Towers ◽  
Carlos Maluquer de Motes
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Immune Activation ◽  
Cellular Mechanism ◽  
Innate Immune ◽  
Dna Sensing ◽  
Dna Viruses ◽  
Pathogen Invasion ◽  
Double Stranded Dna ◽  
Wide Range

ABSTRACT Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerizes and translocates from the endoplasmic reticulum (ER) to a perinuclear region to mediate IRF-3 activation. Poxviruses are double-stranded DNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here, we investigated the activation of innate immune signaling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerized and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerization and phosphorylation during infection and in response to transfected DNA and cyclic GMP-AMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence. IMPORTANCE Poxviruses are double-stranded DNA viruses infecting a wide range of vertebrates and include the causative agent of smallpox (variola virus) and its vaccine vaccinia virus (VACV). Despite smallpox eradication VACV remains of interest as a therapeutic. Attenuated strains are popular vaccine candidates, whereas replication-competent strains are emerging as efficient oncolytics in virotherapy. The successful therapeutic use of VACV depends on a detailed understanding of its ability to modulate host innate immune responses. DNA sensing is a critical cellular mechanism for pathogen detection and activation of innate immunity that is centrally coordinated by the endoplasmic reticulum-resident protein STING. Here, STING is shown to mediate immune activation in response to MVA, but not in response to virulent VACV strains or other virulent poxviruses, which prevent STING activation and DNA sensing during infection and after DNA transfection. These results provide new insights into poxvirus immune evasion and have implications in the rational design of VACV-based therapeutics.

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 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.

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