scholarly journals DNase treatment improves viral enrichment in agricultural soil viromes

2021 ◽  
Author(s):  
Jackson W Sorensen ◽  
Joanne B. Emerson ◽  
Laura A Zinke ◽  
Anneliek M ter Horst ◽  
Christian Santos-Medellín ◽  
...  
Keyword(s):  
Size Fraction ◽  
Frozen Storage ◽  
Viral Diversity ◽  
Viral Dna ◽  
Read Mapping ◽  
Resultant Data ◽  
Dnase Treatment ◽  
Ecological Patterns ◽  
Viral Communities ◽  
Microbial Dna

The small genomes of most viruses make it difficult to fully capture viral diversity in metagenomes dominated by DNA from cellular organisms. Viral size-fraction metagenomics (viromics) protocols facilitate enrichment of viral DNA from environmental samples, and these protocols typically include a DNase treatment of the post-0.2 μm viromic fraction to remove contaminating free DNA prior to virion lysis. However, DNase may also remove desirable viral genomic DNA (e.g., contained in virions compromised due to frozen storage or laboratory processing), suggesting that DNase-untreated viromes might be useful in some cases. In order to understand how virome preparation with and without DNase treatment influences the resultant data, here we compared 15 soil viromes (7 DNase-treated, 8 untreated) from 8 samples collected from agricultural fields prior to tomato planting. DNase-treated viromes yielded significantly more assembled viral contigs, contained significantly less non-viral microbial DNA, and recovered more viral populations (vOTUs) through read mapping. However, DNase-treated and untreated viromes were statistically indistinguishable, in terms of ecological patterns across viral communities. Although results suggest that DNase treatment is preferable where possible, in comparison to previously reported total metagenomes from the same samples, both DNase-treated and untreated viromes were significantly enriched in viral signatures by all metrics compared, including a ~225 times greater proportion of viral reads in untreated viromes compared to total metagenomes. Thus, even without DNase treatment, viromics was preferable to total metagenomics for capturing viral diversity in these soils, suggesting that preparation of DNase-untreated viromes can be worthwhile when DNase treatment is not possible

scholarly journals DNase Treatment Improves Viral Enrichment in Agricultural Soil Viromes

mSystems ◽  
2021 ◽  
Author(s):  
Jackson W. Sorensen ◽  
Laura A. Zinke ◽  
Anneliek M. ter Horst ◽  
Christian Santos-Medellín ◽  
Alena Schroeder ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Agricultural Soil ◽  
Size Fraction ◽  
Viral Origin ◽  
Popular Method ◽  
Free Dna ◽  
Dnase Treatment ◽  
Viral Communities

Viromics is becoming an increasingly popular method for characterizing soil viral communities. DNase treatment of the viral size fraction prior to DNA extraction is meant to reduce contaminating free DNA and is a common step within viromics protocols to ensure that sequences are of viral origin.

scholarly journals Murine Cytomegalovirus Capsid Assembly Is Dependent on US22 Family Gene M140 in Infected Macrophages

2009 ◽  
Vol 83 (15) ◽  
pp. 7449-7456 ◽  
Author(s):  
Laura K. Hanson ◽  
Jacquelyn S. Slater ◽  
Victoria J. Cavanaugh ◽  
William W. Newcomb ◽  
Lisa L. Bolin ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Early Gene ◽  
Murine Cytomegalovirus ◽  
Genome Replication ◽  
Microtubule Organizing Center ◽  
Viral Dna ◽  
Dnase Treatment ◽  
Organizing Center ◽  
Significant Impairment ◽  
The Stability

ABSTRACT Macrophages are an important target cell for infection with cytomegalovirus (CMV). A number of viral genes that either are expressed specifically in this cell type or function to optimize CMV replication in this host cell have now been identified. Among these is the murine CMV (MCMV) US22 gene family member M140, a nonessential early gene whose deletion (RVΔ140) leads to significant impairment in virus replication in differentiated macrophages. We have now determined that the defect in replication is at the stage of viral DNA encapsidation. Although the rate of RVΔ140 genome replication and extent of DNA cleavage were comparable to those for revertant virus, deletion of M140 resulted in a significant reduction in the number of viral capsids in the nucleus, and the viral DNA remained sensitive to DNase treatment. These data are indicative of incomplete virion assembly. Steady-state levels of both the major capsid protein (M86) and tegument protein M25 were reduced in the absence of the M140 protein (pM140). This effect may be related to the localization of pM140 to an aggresome-like, microtubule organizing center-associated structure that is known to target misfolded and overexpressed proteins for degradation. It appears, therefore, that pM140 indirectly influences MCMV capsid formation in differentiated macrophages by regulating the stability of viral structural proteins.

scholarly journals Fecal Viral Diversity of Captive and Wild Tasmanian Devils Characterized Using Virion-Enriched Metagenomics and Metatranscriptomics

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Rowena Chong ◽  
Mang Shi ◽  
Catherine E. Grueber ◽  
Edward C. Holmes ◽  
Carolyn J. Hogg ◽  
...  
Keyword(s):  
Endangered Species ◽  
Population Decline ◽  
Significant Risk ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Viral Diversity ◽  
Hemorrhagic Disease ◽  
Tasmanian Devil ◽  
Dna Viruses ◽  
Viral Communities ◽  
Devil Facial Tumor Disease

ABSTRACT The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumor disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and metatranscriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from two captive and four wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses, and a gammaherpesvirus, were identified, as well as known mammalian pathogens such as rabbit hemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with metatranscriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and metatranscriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and metatranscriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample. IMPORTANCE The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumor disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (dasyurid herpesvirus 2 [DaHV-2]), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than in wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and metatranscriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.

scholarly journals Methods for Extraction and Detection of Bacteriophage DNA from the Sputum of Patients with Cystic Fibrosis

2020 ◽  
Author(s):  
Elizabeth B Burgener ◽  
Patrick R Secor ◽  
Michael C Tracy ◽  
Johanna M Sweere ◽  
Elisabeth M Bik ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Clinical Samples ◽  
Proof Of Concept ◽  
Chronic Infections ◽  
Optimized Protocol ◽  
Airway Infections ◽  
Viral Communities ◽  
Microbial Dna ◽  
The Impact ◽  
Specific Bacteriophage

AbstractThere is increasing interest in the pulmonary microbiome’s bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains challenging. Here, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with a particular emphasis on detecting bacteriophage viron-derived nucleic acid. We evaluate the impact of bead-beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum as a proof of concept. Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for PCR or sequencing-based studies. These studies will facilitate future assessments of bacteriophage and bacteria in sputum from patients with CF.Author SummaryPatients with cystic fibrosis (CF) have thickened secretions and develop chronic infections in their airways. While we culture bacterial pathogens from expectorated sputum this method favors detection of certain organisms. There is greater understanding that the microbiome with in the airway of patients with cystic fibrosis is varied and contains more than just the bacteria that we selectively culture. Processing sputum is difficult as it is very thick. There are many different ways to process sputum depending on what aspect of the sputum is to be studied. We have an interest in a specific bacteriophage, or a virus that infects bacteria. We sought to find the best method to take sputum from a CF patient and extract DNA so that we could detect both bacteria and bacteriophage DNA. We compared different methods that included different combinations of heat, mechanical homogenization, chemical lysis and DNA extraction by commercially available kits. We describe the method we found easiest to execute and produced the best yield for detection of both bacteria and bacteriophage. Our purpose of publishing this method in detail is to facilitate further study of viral and bacterial communities in the sputum of patients with CF.

scholarly journals Minnesota peat viromes reveal terrestrial and aquatic niche partitioning for local and global viral populations

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Anneliek M. ter Horst ◽  
Christian Santos-Medellín ◽  
Jackson W. Sorensen ◽  
Laura A. Zinke ◽  
Rachel M. Wilson ◽  
...  
Keyword(s):  
Protein Content ◽  
Niche Partitioning ◽  
Size Fraction ◽  
Species Boundaries ◽  
Viral Community ◽  
Ecological Patterns ◽  
Significant Enrichment ◽  
Host Ecology ◽  
Depth Water ◽  
Surface Peat

Abstract Background Peatlands are expected to experience sustained yet fluctuating higher temperatures due to climate change, leading to increased microbial activity and greenhouse gas emissions. Despite mounting evidence for viral contributions to these processes in peatlands underlain with permafrost, little is known about viruses in other peatlands. More generally, soil viral biogeography and its potential drivers are poorly understood at both local and global scales. Here, 87 metagenomes and five viral size-fraction metagenomes (viromes) from a boreal peatland in northern Minnesota (the SPRUCE whole-ecosystem warming experiment and surrounding bog) were analyzed for dsDNA viral community ecological patterns, and the recovered viral populations (vOTUs) were compared with our curated PIGEON database of 266,125 vOTUs from diverse ecosystems. Results Within the SPRUCE experiment, viral community composition was significantly correlated with peat depth, water content, and carbon chemistry, including CH4 and CO2 concentrations, but not with temperature during the first 2 years of warming treatments. Peat vOTUs with aquatic-like signatures (shared predicted protein content with marine and/or freshwater vOTUs) were significantly enriched in more waterlogged surface peat depths. Predicted host ranges for SPRUCE vOTUs were relatively narrow, generally within a single bacterial genus. Of the 4326 SPRUCE vOTUs, 164 were previously detected in other soils, mostly peatlands. None of the previously identified 202,371 marine and freshwater vOTUs in our PIGEON database were detected in SPRUCE peat, but 0.4% of 80,714 viral clusters (VCs, grouped by predicted protein content) were shared between soil and aquatic environments. On a per-sample basis, vOTU recovery was 32 times higher from viromes compared with total metagenomes. Conclusions Results suggest strong viral “species” boundaries between terrestrial and aquatic ecosystems and to some extent between peat and other soils, with differences less pronounced at higher taxonomic levels. The significant enrichment of aquatic-like vOTUs in more waterlogged peat suggests that viruses may also exhibit niche partitioning on more local scales. These patterns are presumably driven in part by host ecology, consistent with the predicted narrow host ranges. Although more samples and increased sequencing depth improved vOTU recovery from total metagenomes, the substantially higher per-sample vOTU recovery after viral particle enrichment highlights the utility of soil viromics.

scholarly journals A Review of Marine Viruses in Coral Ecosystem

2021 ◽  
Vol 9 (7) ◽  
pp. 711
Author(s):  
Logajothiswaran Ambalavanan ◽  
Shumpei Iehata ◽  
Rosanne Fletcher ◽  
Emylia H. Stevens ◽  
Sandra C. Zainathan
Keyword(s):  
Marine Invertebrates ◽  
Marine Sponges ◽  
Detection Methods ◽  
Viral Diversity ◽  
Double Stranded Rna ◽  
Identification Methods ◽  
Double Stranded Dna ◽  
Marine Viruses ◽  
Coral Reef Ecosystems ◽  
Viral Communities

Coral reefs are among the most biodiverse biological systems on earth. Corals are classified as marine invertebrates and filter the surrounding food and other particles in seawater, including pathogens such as viruses. Viruses act as both pathogen and symbiont for metazoans. Marine viruses that are abundant in the ocean are mostly single-, double stranded DNA and single-, double stranded RNA viruses. These discoveries were made via advanced identification methods which have detected their presence in coral reef ecosystems including PCR analyses, metagenomic analyses, transcriptomic analyses and electron microscopy. This review discusses the discovery of viruses in the marine environment and their hosts, viral diversity in corals, presence of virus in corallivorous fish communities in reef ecosystems, detection methods, and occurrence of marine viral communities in marine sponges.

scholarly journals Human land-use impacts viral diversity and abundance in a New Zealand river

2022 ◽  
Author(s):  
Rebecca French ◽  
Justine Charon ◽  
Callum Le Lay ◽  
Chris Muller ◽  
Edward C Holmes
Keyword(s):  
Land Use ◽  
Water Systems ◽  
Viral Diversity ◽  
Native Forest ◽  
Virus Species ◽  
Human Land Use ◽  
Forest Sites ◽  
Viral Communities ◽  
The Impact ◽  
Urban Sites

Although water borne viruses have important implications for the health of humans and other animals, little is known about the impact of human land use on viral diversity and evolution in water systems such as rivers. We used metagenomic next generation sequencing to compare the diversity and abundance of viruses at sampling sites along a single river in New Zealand that differed in human land use impact, ranging from pristine to urban. From this we identified 504 putative virus species, of which 97% were novel. Many of the novel viruses were highly divergent, and likely included a new subfamily within the Parvoviridae. We identified at least 63 virus species that may infect vertebrates, most likely fish and water birds, from the Astroviridae, Birnaviridae, Parvoviridae and Picornaviridae. No putative human viruses were detected. Importantly, we observed differences in the composition of viral communities at sites impacted by human land use (farming and urban) compared to native forest sites (pristine). At the viral species level, the urban sites had higher diversity (327 virus species) than the farming (n=150) and pristine sites (n=119), and more viruses were shared between the urban and farming sites (n=76) than between the pristine and farming or urban sites (n=24). The two farming sites had a lower viral abundance across all host types, while the pristine sites had a higher abundance of viruses associated with animals, plants and fungi. We also identified viruses linked to agriculture and human impact at the river sampling sites in farming and urban areas that were not present at the native forest sites. Overall, our study shows that human land use can impact viral communities in rivers, such that further work is needed to reduce the impact of intensive farming and urbanization on water systems.

scholarly journals The VirAnnot Pipeline: A Resource for Automated Viral Diversity Estimation and Operational Taxonomy Units Assignation for Virome Sequencing Data

2019 ◽  
Vol 3 (4) ◽  
pp. 256-259 ◽  
Author(s):  
Marie Lefebvre ◽  
Sébastien Theil ◽  
Yuxin Ma ◽  
Thierry Candresse
Keyword(s):  
High Throughput Sequencing ◽  
International Committee ◽  
Viral Metagenomics ◽  
Viral Diversity ◽  
Sequencing Data ◽  
Automated Identification ◽  
Protein Motifs ◽  
Identity Threshold ◽  
Viral Communities ◽  
Clustering Approach

Viral metagenomics relies on high-throughput sequencing and on bioinformatic analyses to access the genetic content and diversity of entire viral communities. No universally accepted strategy or tool currently exists to define operational taxonomy units (OTUs) and evaluate viral alpha or beta diversity from virome data. Here we present a new bioinformatic resource, the VirAnnot (automated viral diversity estimation) pipeline, which performs the automated identification of OTUs. Reverse-position-specific BLAST (RPS-Blastn) is used to detect conserved viral protein motifs. The corresponding contigs are then aligned and a clustering approach is used to group in the same OTU contigs sharing more than a set identity threshold. A 10% threshold has been validated as producing OTUs that reasonably approach, in many families, the International Committee for the Taxonomy of Viruses taxonomy and can therefore be used as a proxy to viral species.

scholarly journals Viromes outperform total metagenomes in revealing the spatiotemporal patterns of agricultural soil viral communities

2021 ◽  
Author(s):  
Christian Santos-Medellin ◽  
Laura A. Zinke ◽  
Anneliek M. ter Horst ◽  
Danielle L. Gelardi ◽  
Sanjai J. Parikh ◽  
...  
Keyword(s):  
Community Dynamics ◽  
Agricultural Soils ◽  
Size Fraction ◽  
Growing Season ◽  
Taxonomic Diversity ◽  
Soil Heterogeneity ◽  
Decay Rates ◽  
Spatial Gradient ◽  
Microbial Community Dynamics ◽  
Viral Communities

AbstractViruses are abundant yet understudied members of soil environments that influence terrestrial biogeochemical cycles. Here, we characterized the dsDNA viral diversity in biochar-amended agricultural soils at the preplanting and harvesting stages of a tomato growing season via paired total metagenomes and viral size fraction metagenomes (viromes). Size fractionation prior to DNA extraction reduced sources of nonviral DNA in viromes, enabling the recovery of a vaster richness of viral populations (vOTUs), greater viral taxonomic diversity, broader range of predicted hosts, and better access to the rare virosphere, relative to total metagenomes, which tended to recover only the most persistent and abundant vOTUs. Of 2961 detected vOTUs, 2684 were recovered exclusively from viromes, while only three were recovered from total metagenomes alone. Both viral and microbial communities differed significantly over time, suggesting a coupled response to rhizosphere recruitment processes and/or nitrogen amendments. Viral communities alone were also structured along an 18 m spatial gradient. Overall, our results highlight the utility of soil viromics and reveal similarities between viral and microbial community dynamics throughout the tomato growing season yet suggest a partial decoupling of the processes driving their spatial distributions, potentially due to differences in dispersal, decay rates, and/or sensitivities to soil heterogeneity.

scholarly journals Minnesota peat viromes reveal terrestrial and aquatic niche partitioning for local and global viral populations

2020 ◽  
Author(s):  
Anneliek M ter Horst ◽  
Christian Santos-Medellin ◽  
Jackson W Sorensen ◽  
Laura A Zinke ◽  
Rachel M Wilson ◽  
...  
Keyword(s):  
Niche Partitioning ◽  
Size Fraction ◽  
Species Boundaries ◽  
Genus Level ◽  
Viral Community ◽  
Ecological Patterns ◽  
Significant Enrichment ◽  
Host Ecology ◽  
Depth Water ◽  
Surface Peat

Background: Peatlands are expected to experience sustained yet fluctuating higher temperatures due to climate change, leading to increased microbial activity and greenhouse gas emissions. Despite mounting evidence for viral contributions to these processes in peatlands underlain with permafrost, little is known about viruses in other peatlands. More generally, soil viral biogeography and its potential drivers are poorly understood at both local and global scales. Here, 87 metagenomes and five viral size-fraction metagenomes (viromes) from a boreal peatland in northern Minnesota (the SPRUCE whole-ecosystem warming experiment and surrounding bog) were analyzed for dsDNA viral community ecological patterns, and the recovered viral populations (vOTUs) were compared to our curated PIGEON database of 266,805 vOTUs from diverse ecosystems. Results: Within the SPRUCE experiment, viral community composition was significantly correlated with peat depth, water content, and carbon chemistry, including CH4 and CO2 concentrations, but not with temperature during the first two years of warming treatments. Peat vOTUs with aquatic-like signatures (shared predicted protein content with marine and/or freshwater vOTUs) were significantly enriched in more waterlogged surface peat depths. Predicted host ranges for SPRUCE vOTUs were relatively narrow, generally within a single bacterial genus. Of the 4,326 SPRUCE vOTUs, 164 were previously detected in other soils, mostly peatlands. None of the previously identified 202,372 marine and freshwater vOTUs in our PIGEON database were detected in SPRUCE peat, but 1.9% of 78,203 genus-level viral clusters (VCs) were shared between soil and aquatic environments. On a per-sample basis, vOTU recovery was 32 times higher from viromes compared to total metagenomes. Conclusions: Results suggest strong viral "species" boundaries between terrestrial and aquatic ecosystems and to some extent between peat and other soils, with differences less pronounced at the "genus" level. The significant enrichment of aquatic-like vOTUs in more waterlogged peat suggests that viruses may also exhibit niche partitioning on more local scales. These patterns are presumably driven in part by host ecology, consistent with the predicted narrow host ranges. Although more samples and increased sequencing depth improved vOTU recovery from total metagenomes, the substantially higher per-sample vOTU recovery after viral particle enrichment highlights the utility of soil viromics.

Sign in / Sign up

Export Citation Format

Share Document