Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

Rosseel, Toon; Ozhelvaci, Orkun; Freimanis, Graham; Borm, Steven Van

doi:10.1016/j.jviromet.2015.05.010

Cited by 48 publications

(57 citation statements)

References 47 publications

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“…The nature of the sequencing protocol implies limited amplification of the viral genetic material, and a significant competition from the larger human genome. Therefore, this approach may not identify lower concentration viruses that could be revealed by using viral particles enrichment [58, 59] or viral genome capture [60, 61]. The latter methods rest on the ability to capture closely related sequences by hybridization to short conserved probes.…”

Section: Discussionmentioning

confidence: 99%

The blood DNA virome in 8,000 humans

Moustafa¹,

Xie²,

Kirkness³

et al. 2017

PLoS Pathog

257

245

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The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

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Section: Discussionmentioning

confidence: 99%

The blood DNA virome in 8,000 humans

Moustafa¹,

Xie²,

Kirkness³

et al. 2017

PLoS Pathog

257

245

View full text Add to dashboard Cite

show abstract

“…During autopsy, a swollen liver and spleen were detected as sole macroscopic lesions. The brain tissue was homogenized in phosphate-buffered saline (10% [wt/vol]), pretreated by 0.45-µM-pore-size selective filtration and nuclease, and RNA was extracted as previously described (4, 5). cDNA was synthesized using SuperScript IV reverse transcriptase (Thermo Fisher Scientific) and random hexamer primers, according to the manufacturer’s instructions, followed by double-strand cDNA synthesis using the NEBNext mRNA second-strand synthesis module (New England BioLabs), according to the manufacturer’s instructions.…”

Section: Genome Announcementmentioning

confidence: 99%

Complete Coding Sequence of Usutu Virus Strain Gracula religiosa/U1609393/Belgium/2016 Obtained from the Brain Tissue of an Infected Captive Common Hill Myna ( Gracula religiosa )

et al. 2017

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“…To date, if the required sample volume is large and a pathogen needs to be enriched for detection, the conventional sample preparation assays use immunological capture or physical separation (Rosseel et al, 2015;Yeh et al, 2014). Immunological capture requires antibodies based on previous knowledge of the targets and it therefore limited when identifying new or emerging virus strains.…”

Section: Introductionmentioning

confidence: 99%

“…DMA and DMP are known as crosslinking reagents and contain two imidoester groups, which react with amine groups of nucleic acids, proteins and cells to form amidine bonds Shin et al, 2015). HI reagents including dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP) and dimethyl suberimidate (DMS) with assembled double microfluidic disposable chips (ADC) are used to effectively capture pathogens (bacteria and viruses) from the clinical samples within 10 min.…”

Section: Introductionmentioning

confidence: 99%

Simple and label-free pathogen enrichment via homobifunctional imidoesters using a microfluidic (SLIM) system for ultrasensitive pathogen detection in various clinical specimens

Jin

Koo

Lee

et al. 2018

Biosensors and Bioelectronics

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Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.

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Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

Cited by 48 publications

References 47 publications

The blood DNA virome in 8,000 humans

The blood DNA virome in 8,000 humans

Complete Coding Sequence of Usutu Virus Strain Gracula religiosa/U1609393/Belgium/2016 Obtained from the Brain Tissue of an Infected Captive Common Hill Myna ( Gracula religiosa )

Simple and label-free pathogen enrichment via homobifunctional imidoesters using a microfluidic (SLIM) system for ultrasensitive pathogen detection in various clinical specimens

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