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Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

ABSTRACT: High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest. A viral vaccine crude harvest and a cell substrate matrix were spiked with 22 viruses. Specificity was demonstrated for all 22 viruses at the species level. Our method was capable of detecting and identifying adventitious viruses spiked at 10⁴ genome copies per milliliter in a viral vaccine crude harvest and 0.01 viral genome copies spiked per cell in a cell substrate matrix. Moreover, 9 of the 11 NIH model viruses with published in vivo data were detected by HTS with an equivalent or better sensitivity (in a viral vaccine crude harvest). Our general-purpose HTS method is unbiased and highly sensitive for the detection of adventitious viruses, and has a large breadth of detection, which may obviate the need to perform in vivo testing.

SUBMITTER: Charlebois RL

PROVIDER: S-EPMC7368052 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

Charlebois Robert L RL Sathiamoorthy Sarmitha S Logvinoff Carine C Gisonni-Lex Lucy L Mallet Laurent L Ng Siemon H S SHS

NPJ vaccines 20200717 1

High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest. A viral vaccine crude harvest and a cell substrate matrix were spiked ...[more]

PMID: 32699651

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Project description:The 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.IMPORTANCE The 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.

Project description: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.

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Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

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Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection.

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