Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids

ABSTRACTThe use of high-throughput sequencing (HTS) to identify viruses in biologicals differs from current molecular approaches, since its use enables an unbiased approach to detection without the need to design specific primers to preamplify target sequences. Its broad range of detection and analytical sensitivity make it an important tool to ensure that biologicals are free from adventitious viruses. Similar to other molecular methods, however, identification of viral sequences in cells by HTS does not prove viral infection, since this could reflect carryover of inert viral sequences from reagents or other sources or the presence of transcriptionally inactive cellular sequences. Due to the broad range of detection associated with HTS, the above can potentially be perceived as a drawback for the testing of pharmaceutical biological products using this method. In order to avoid the identification of inert viral sequences, we present a methodology based on metabolic RNA labeling and sequencing, which enables the specific identification of newly synthesized viral RNAs in infected cells, resulting in the ability to unambiguously distinguish active infection by DNA or RNA viruses from inert nucleic acids. In the present study, we report the ability to differentiate Vero cells acutely infected by a single-stranded positive-sense RNA virus (tick-borne encephalitis virus) from cells which have been in contact with nonreplicating virus particles. Additionally, we also found a laboratory contamination by the squirrel monkey retrovirus of our Vero cell line, which was derived from an Old World (African green) monkey, a type of contamination which until now has been identified only in cells derived from primates from the New World.IMPORTANCEThe use of high-throughput sequencing (HTS) to identify viral contamination of biological products is extremely sensitive and provides a broad range of detection. Nevertheless, viral sequences identified can also be inert. Examples include contamination resulting from reagents or the presence of inactivated viruses in animal-derived components of the cell culture medium. We therefore developed a method that relies on the sequencing of newly synthesized RNAs, an unequivocal sign of the presence of a transcriptionally active virus. This improvement in the specificity of viral testing increases the acceptability of HTS as a standard test for cells used in manufacturing biologicals and in biotherapies.

Download Full-text

Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation

Viruses ◽

10.3390/v10100566 ◽

2018 ◽

Vol 10 (10) ◽

pp. 566 ◽

Cited By ~ 9

Author(s):

Siemon Ng ◽

Cassandra Braxton ◽

Marc Eloit ◽

Szi Feng ◽

Romain Fragnoud ◽

...

Keyword(s):

Sample Preparation ◽

Nucleic Acids ◽

High Throughput ◽

High Throughput Sequencing ◽

Virus Detection ◽

Extraction Methods ◽

Control Measures ◽

Acid Extraction ◽

Library Preparation ◽

Sample Processing

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

Download Full-text

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection

mSphere ◽

10.1128/mspheredirect.00069-18 ◽

2018 ◽

Vol 3 (2) ◽

Cited By ~ 70

Author(s):

Norman Goodacre ◽

Aisha Aljanahi ◽

Subhiksha Nandakumar ◽

Mike Mikailov ◽

Arifa S. Khan

Keyword(s):

High Throughput ◽

Selection Criteria ◽

High Throughput Sequencing ◽

Bioinformatics Analysis ◽

Virus Detection ◽

Sequence Diversity ◽

Endogenous Retroviruses ◽

Viral Sequence ◽

Novel Virus ◽

Viral Sequences

ABSTRACTDetection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank.IMPORTANCETo facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection.

Download Full-text

A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection

mSphere ◽

10.1128/msphere.00307-17 ◽

2017 ◽

Vol 2 (5) ◽

Cited By ~ 10

Author(s):

Arifa S. Khan ◽

Siemon H. S. Ng ◽

Olivier Vandeputte ◽

Aisha Aljanahi ◽

Avisek Deyati ◽

...

Keyword(s):

Sample Preparation ◽

Cell Line ◽

High Throughput ◽

Future Development ◽

High Throughput Sequencing ◽

Collaborative Study ◽

Virus Detection ◽

Biological Products ◽

Reference Virus ◽

Complex Technology

ABSTRACT Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms. The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. IMPORTANCE Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms.

Download Full-text

Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis

mBio ◽

10.1128/mbio.01491-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 150

Author(s):

Thomas Briese ◽

Amit Kapoor ◽

Nischay Mishra ◽

Komal Jain ◽

Arvind Kumar ◽

...

Keyword(s):

Nucleic Acids ◽

High Throughput ◽

High Throughput Sequencing ◽

Viral Infections ◽

Limit Of Detection ◽

Virus Detection ◽

Fold Increase ◽

Sequencing Platform ◽

Oligonucleotide Library ◽

Capture Sequencing

ABSTRACT Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics.IMPORTANCEVirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.

Download Full-text

Comparison of high throughput sequencing to standard protocols for virus detection in berry crops

Plant Disease ◽

10.1094/pdis-05-21-0949-re ◽

2021 ◽

Author(s):

Dan Edward Veloso Villamor ◽

Karen E Keller ◽

Robert Martin ◽

Ioannis Emmanouil Tzanetakis

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Wide Spectrum ◽

Virus Detection ◽

Rt Pcr ◽

Host Interactions ◽

Growing Seasons ◽

Berry Crops ◽

Sampling Times ◽

Better Than

A comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium and 8 Rubus) with known virus profiles was completed. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, RT-PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared to RT-PCR on a wide spectrum of virus variants and discovery of novel viruses. More importantly, in most cases where the two protocols showed parallel virus detection, 11 viruses in 16 berry selections were not consistently detected by both methods at all sampling points. Based on these data we propose a four sampling times/two-year testing requirement for berry and potentially other crops to ensure that no virus remains undetected independent of titer, distribution or other virus/virus or virus/host interactions.

Download Full-text

High Throughput Sequencing For Plant Virus Detection and Discovery

Phytopathology ◽

10.1094/phyto-07-18-0257-rvw ◽

2019 ◽

Vol 109 (5) ◽

pp. 716-725 ◽

Cited By ~ 44

Author(s):

D. E. V. Villamor ◽

T. Ho ◽

M. Al Rwahnih ◽

R. R. Martin ◽

I. E. Tzanetakis

Keyword(s):

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing ◽

Virus Detection ◽

Agricultural Crops ◽

Virus Discovery ◽

Plant Virus Detection ◽

Virus Biology ◽

Disease Causality

Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists’ utility in their research.

Download Full-text

Comprehensive pathogen detection in sera of Kawasaki disease patients by high-throughput sequencing: a retrospective exploratory study

BMC Pediatrics ◽

10.1186/s12887-020-02380-7 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Yuka Torii ◽

Kazuhiro Horiba ◽

Satoshi Hayano ◽

Taichi Kato ◽

Takako Suzuki ◽

...

Keyword(s):

Kawasaki Disease ◽

High Throughput ◽

High Throughput Sequencing ◽

Sequence Data ◽

Early Stage ◽

Rna Virus ◽

Systemic Vasculitis ◽

Human Herpesvirus ◽

Acute Stage ◽

Serum Samples

Abstract Background Kawasaki disease (KD) is an idiopathic systemic vasculitis that predominantly damages coronary arteries in children. Various pathogens have been investigated as triggers for KD, but no definitive causative pathogen has been determined. As KD is diagnosed by symptoms, several days are needed for diagnosis. Therefore, at the time of diagnosis of KD, the pathogen of the trigger may already be diminished. The aim of this study was to explore comprehensive pathogens in the sera at the acute stage of KD using high-throughput sequencing (HTS). Methods Sera of 12 patients at an extremely early stage of KD and 12 controls were investigated. DNA and RNA sequences were read separately using HTS. Sequence data were imported into the home-brew meta-genomic analysis pipeline, PATHDET, to identify the pathogen sequences. Results No RNA virus reads were detected in any KD case except for that of equine infectious anemia, which is known as a contaminant of commercial reverse transcriptase. Concerning DNA viruses, human herpesvirus 6B (HHV-6B, two cases) and Anelloviridae (eight cases) were detected among KD cases as well as controls. Multiple bacterial reads were obtained from KD and controls. Bacteria of the genera Acinetobacter, Pseudomonas, Delfita, Roseomonas, and Rhodocyclaceae appeared to be more common in KD sera than in the controls. Conclusion No single pathogen was identified in serum samples of patients at the acute phase of KD. With multiple bacteria detected in the serum samples, it is difficult to exclude the possibility of contamination; however, it is possible that these bacteria might stimulate the immune system and induce KD.

Download Full-text

Advanced Virus Detection Technologies Interest Group (AVDTIG): Efforts on High Throughput Sequencing (HTS) for Virus Detection

PDA Journal of Pharmaceutical Science and Technology ◽

10.5731/pdajpst.2016.007161 ◽

2016 ◽

Vol 70 (6) ◽

pp. 591-595 ◽

Cited By ~ 12

Author(s):

A. S. Khan ◽

D. A. Vacante ◽

J.-P. Cassart ◽

S. H. S. Ng ◽

C. Lambert ◽

...

Keyword(s):

Interest Group ◽

High Throughput ◽

High Throughput Sequencing ◽

Virus Detection ◽

Detection Technologies

Download Full-text

Evaluation of Three Automated Nucleic Acid Extraction Systems for Identification of Respiratory Viruses in Clinical Specimens by Multiplex Real-Time PCR

BioMed Research International ◽

10.1155/2014/430650 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Yoonjung Kim ◽

Mi-Soon Han ◽

Juwon Kim ◽

Aerin Kwon ◽

Kyung-A Lee

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

False Negative ◽

Virus Detection ◽

Turnaround Time ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Analytical Sensitivity ◽

Nucleic Acid Extraction ◽

Negative Results

A total of 84 nasopharyngeal swab specimens were collected from 84 patients. Viral nucleic acid was extracted by three automated extraction systems: QIAcube (Qiagen, Germany), EZ1 Advanced XL (Qiagen), and MICROLAB Nimbus IVD (Hamilton, USA). Fourteen RNA viruses and two DNA viruses were detected using the Anyplex II RV16 Detection kit (Seegene, Republic of Korea). The EZ1 Advanced XL system demonstrated the best analytical sensitivity for all the three viral strains. The nucleic acids extracted by EZ1 Advanced XL showed higher positive rates for virus detection than the others. Meanwhile, the MICROLAB Nimbus IVD system was comprised of fully automated steps from nucleic extraction to PCR setup function that could reduce human errors. For the nucleic acids recovered from nasopharyngeal swab specimens, the QIAcube system showed the fewest false negative results and the best concordance rate, and it may be more suitable for detecting various viruses including RNA and DNA virus strains. Each system showed different sensitivity and specificity for detection of certain viral pathogens and demonstrated different characteristics such as turnaround time and sample capacity. Therefore, these factors should be considered when new nucleic acid extraction systems are introduced to the laboratory.

Download Full-text